Team:ATOMS-Turkiye/Extras/Notebooke
{{ATOMS-Turkiye/Test}}
NOTEBOOK
December
9.12.-13.12. We filled a survey to become next generation of ATOMS members.
Waiting in excitement and wondering who will be the members of this year’s team.
22.12. Aaaaand the results of the interviews we made are announced! Now we’re a crowded team of 25 people. Let’s see what’ll happen next.
26.12. Had our first meeting and our first topic was: sponsorship.
January
We had training classes at 08.01-10.01. January. Now it is time to learn what iGEM wants from us.
10.01.-23.01. We made researches about previous iGEM teams, wondering who did what.
23.01.-28.01. Hooray! It’s holiday! Home sweet home.
It’s time to find our own project. After now, we started to have meetings every Monday and Thursday, becase we need to work a lot for finding a new project. Also, still trying to find sponsors.
February
01.02. As we are ATOMS, those Sundays needed to be filled too! We made subgroups in our team and started to search previous Grand Prized-Teams. Now every group will present a team at the meetings.
We have just built ATOMS gene library.
02.02. We decided to attend some competitions due to the lack of sponsors. We attended ‘illnesses with imagery’. Talented people is a must in a team.
13.02. Drawings were done and sent. We believe we will be successful. We are still doing researches in full swing.
March
05.03. Today they filmed our school, and our laboratory so. We had so much fun while they were shooting us!
11.03. The art competiton’s results are just announced. Disappointment. There was not even a winner of first place. We convinced ourselves with thinking they don’t have a sense of art.
14.03. We made a biobrcik design workshop till we see the sunlight! At the late times of night, every group explained their biobrick designs. Now everyone knows what a promoter is :)
17.03. Lab-cleaning party! We don’t wanna be contaminated.
April
05.04. We bid farewell to our teammate Furkan Beştepe. He went to USA.
10.04. Happy birthday Gülnihal! By the way your birthday cake was yummy.
12.04. Daytime wasn’t enough so we started to have meetings in the nights! After working a lot, we deserved eating a delicious meal. Today we also started to search about Toehold.
13.04. We are still learnig about Toehold. It has a potential to be used.
27.04. Fun at the lab.
30.04. ATOMS Ladieas worked on a universal blood idea till the morning.
May
03.05. We must hurry up to find a project. Camp time at Asya Thermal Hotel.
04.05. Why there isn’t anyone in the lab?
06.05. Finally found our project idea! ULCER AND CANCER
09.05. Perfecting our cancer switch system.
15.05. Presented our project to the dean Mehmet Gündüz and the whole genetic department of our faculty! He treated us with Turkish tea and snacks.
24.05. We held a meet-up with other Turkish iGEM teams! Time to make some presentations.
June
This month we designed our genes and studied for our exams at the same time. In short it was a tough week.
Week 1
57-64˚C
Results weren’t matching with expected results, experiment will be repeated.
{| border="1" class="wikitable" !Gradient PCR from pCAGGS |- |||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||16.3 ul||2.0 ul||25.0 ul |- |9X||22.5 ul||22.5 ul||4.5 ul||4.5 ul||4.5 ul||1.8 ul||146.7 ul||18.0 ul||225.0 ul |}
52-59˚C
Results weren’t matching with expected results, experiment will be repeated.
July
01.07. Some preparetions were made for experiments.
05.07. Today is the first day of shooting. We just learned picking costumes isn’t as easy as we think.
06.07. If there is a film, then there is After Effects work to do. Good luck with this, dear teammate Şahika.
08.07. We made a presentation to the pre-med students which came from USA for intership and they loved our project.
21.07. Drawing pictures for wiki began. Thus we discovered our teammate Kevser’s hidden talents.
22.07. Our gene blocks has just arrived. Let the experiments begin! We are all ready now.
29.07. We released our first video of Virtual Hospital. Also arm’s design is finished.
60-68˚C
Results weren’t matching with expected results, experiment will be repeated.
{| border="1" class="wikitable" !Gradient PCR from pCAGGS (CAG FWD – Chicken β Aktin REVERSE) |- |||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||16.3 ul||2.0 ul||25.0 ul |- |9X||22.5 ul||22.5 ul||4.5 ul||4.5 ul||4.5 ul||1.8 ul||146.7 ul||18.0 ul||225.0 ul |}
60-68˚C
Results weren’t matching with expected results, experiment will be repeated.
Protocols of „Phusion Pol, and Q5 Polymerase” (02.07.2015) {| border="1" class="wikitable" !Phusion DNA Polymerase |- |||dNTP||Fwd primer||Rev primer||Buffer||Phusion Pol.||ddH₂O||DNA||Total |- |1x||0.4 ul||2.0 ul||2.0 ul||4.0 ul||0.2 ul||10.4 ul||1.0 ul||20.0 ul |} {| border="1" class="wikitable" !Cycling |- |||98˚C||98˚C||56˚C||72˚C||72˚C||Cycle |- |Time||2’||10’’||30’’||1’||5’||35x |}
{| border="1" class="wikitable" !Q5 DNA Polymerase |- |||dNTP||Fwd primer||Rev primer||Buffer||Q5 Pol.||ddH₂O||DNA||Total |- |1x||0.5 ul||2.5 ul||2.5 ul||5.0 ul||0.25 ul||8.25 ul||1.0 ul||20.0 ul |} {| border="1" class="wikitable" !Cycling |- |||98˚C||98˚C||56˚C||72˚C||72˚C||Cycle |- |Time||2’||10’’||30’’||1’||5’||35x |}
Defterde jel görüntüsü yok.
62-64˚C
Result: Gel extraction was performed. PCR (+): 680 bp
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||eb||biospec||7/8/2015 1:56:43 AM||0.1||ng/ul||0.002||-0.009||-0.25||-0.19||DNA||50.00 |- |2||pCAG||biospec||7/8/2015 1:58:14 AM||13.7||ng/ul||0.275||0.138||1.98||0.15||DNA||50.00 |}
Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” {| border="1" class="wikitable" !Digestion of “pTRE” and “Promoter pCAG” |- |||pTRE (1536 ng/ul)||pCAG (promoter) (13.7 ng/ul)||EcoRI||XhoI ||Neb 3.1 Buffer||ddH₂O||Total |- |1||3.2 ul||-||0.5 ul||0.5 ul||2.0 ul||13.8 ul||20.0 ul |- |2||-||10.0 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul |}
pTRE-delta-TRE was made after digestion.
Concentration: 99.9 ng/ul
The final concentration of pCAG is 6.855 ng/ul.
{| border="1" class="wikitable" !Ligation of „pTRE TRE“ and „Digested promoter pCAG“ |- | |- |||pTRE TRE |- |pCAG ||T4 DNA Ligase||Buffer||ddH₂O||Total|| |- |1:1||3.0 ul||7.0 ul||0.5 ul||2.0 ul||7.5 ul||20.0 ul|| |}
Ligation products were transformed into E.Coli/BL321 strain.
Result: No colonies were observed.
Digestion of “pTEToff and pET45 Vectors” {| border="1" class="wikitable" !Digestion of “pTEToff and pET45 Vectors” |- |||pTEToff (1141 ng/ul)||pET45 (485 ng/ul)||XhoI (Neb)||BamHI (Neb)||SalI (Thermo)||HindIII (Thermo)||Cut Smart Buffer||Fast Digest Buffer||ddH₂O||Total|| |- |1||3.1 ul||-||-||-||0.5 ul||0.5 ul||-||2.0 ul||13.9 ul||20.0 ul||37˚C 1h |- |2||-||4.1 ul||0.5 ul||0.5 ul||-||-||2.0 ul||-||12.9 ul||20.0 ul||37˚C 2h |}
Result: Bands were at the expected section. Gel extraction was made.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||eb||biospec||7/10/2015 11:37:54 PM||-0.4||ng/ul||-0.008||-0.015||0.58||-0.39||DNA||50.00 |- |2||pET45 x+b||biospec||7/10/2015 11:40:59 PM||59.0||ng/ul||1.180||0.612||1.93||0.74||DNA||50.00 |- |3||pTEToff s+h||biospec||7/10/2015 11:41:58 PM||55.5||ng/ul||1.110||0.581||1.91||0.25||DNA||50.00 |}
Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” – Repeat {| border="1" class="wikitable" !Digestion of “pTRE with EcoRI/XhoI” |- |||pTRE||XhoI||EcoRI||Neb 2.1 Buffer||ddH₂O||Total|| |- |1||3.2 ul||0.5 ul||0.5 ul||2.0 ul||13,8 ul||20.0 ul||37˚C 2h |- |2||3.2 ul||0.5 ul||0.5 ul||2.0 ul||13,8 ul||20.0 ul||37˚C 2h/0.5 ul CIP/37˚C 30’/50˚C 30’ |}
Result: Gel extraction was made.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||eb||biospec||7/9/2015 6:32:17 PM||-0.7||ng/ul||-0.015||-0.022||0.66||0.21||DNA||50.00 |- |2||hre cmv mini||biospec||7/9/2015 6:34:10 PM||13.8||ng/ul||0.277||0.141||1.97||0.03||DNA||50.00 |- |3||ptre delta tre cip -||biospec||7/9/2015 6:34:54 PM||88.7||ng/ul||1.774||0.941||1.88||0.24||DNA||50.00 |- |4||ptre delta tre cip +||biospec||7/9/2015 6:35:35 PM||86.0||ng/ul||1.721||0.947||1.82||0.25||DNA||50.00 |}
Creating “Plasmid pCAG” – Continue {| border="1" class="wikitable" !Ligation of „pTRE TRE“ and „Digested promoter pCAG“ |- | |- |||pTRE TRE CIP (+)||pTRE TRE CIP (-)||pCAG (6.85 ng/ul)||T4 DNA Ligase||Buffer||ddH₂O||Total |- |1||3.0 ul||-||7.0 ul||0.5 ul||2.0 ul||7.5 ul||20.0 ul |- |2||-||3.0 ul||7.0 ul||0.5 ul||2.0 ul||7.5 ul||20.0 ul |}
Room Temperature 1h
Result: Transformation was made. No colonies were observed at first plate. At the second plate there was five colonies. Colony PCR will be made.
Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.
(+) bant: 923 bp
(-) bant: 705 bp
Creating “Plasmid pCAG” – Repeat {| border="1" class="wikitable" !Ligation of „pTRE TRE“ and „Digested Promoter pCAG“ |- | |- |||pTRE TRE CIP (+)||pTRE TRE CIP (-)||pCAG (6.85 ng/ul)||T4 DNA Ligase||Buffer||ddH₂O||Total |- |1||3.0 ul||-||2.5 ul||0.5 ul||2.0 ul||12.0 ul||20.0 ul |- |2||-||3.0 ul||2.5 ul||0.5 ul||2.0 ul||12.0 ul||20.0 ul |}
Vector:Insert
3:1
RT 2h
Transformation at BL21.
CIP (+): No colonies were absorved.
CIP (-): Colony PCR will be made.
Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated.
(+) bant: 923 bp
(-) bant: 705 bp
Creating “pCAG (Plasmid) – Repeat {| border="1" class="wikitable" !PCR from “pCAGGS” |- |||dNTP||CAG fwd||CAG rev||Chicken β Akt. Rev||pCAGGS||Phusion Pol||Buffer||ddH₂O||Total |- |1||2.0 ul||10.0 ul||10.0 ul||-||5.0 ul||1.0 ul||20.0 ul||52.0 ul||100.0 ul |- |2||2.0 ul||10.0 ul||-||10.0 ul||5.0 ul||1.0 ul||20.0 ul||52.0 ul||100.0 ul |} {| border="1" class="wikitable" !Cycling |- |||98˚C||98˚C||64/68˚C||72˚C||72˚C||Cycle |- |Time||2’||10’’||30’’||1’||5’||35x |}
Gel Extraction will made.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||eb||biospec||7/23/2015 6:56:38 PM||0.2||ng/ul||0.005||0.005||0.92||0.14||DNA||50.00 |- |2||pcag e||biospec||7/23/2015 6:58:03 PM||42.9||ng/ul||0.858||0.450||1.91||1.97||DNA||50.00 |- |3||pcag y||biospec||7/23/2015 6:58:53 PM||23.3||ng/ul||0.466||0.233||2.00||1.94||DNA||50.00 |}
Resuspension of “Newly Arrived G-Blocks from IDT”
100 ul TE for all tubes.
{| border="1" class="wikitable" !!!!!ng!!fmol!!TE ul!!fmol/ul!!ul of inserts for 75 fmol |- |1||Toehold for cola||1000||1523||100||15.23||4.92449 |- |2||TnrA-pTnrA-RFP||1000||1032||100||10.32||7.26744 |- |3||ColA-KanR-dTer||1000||816||100||8.16||9.19118 |- |4||HNS for PET||500||1578||100||15.78||4.75285 |- |5||HNS-T108I for PET||500||1578||100||15.78||4.75285 |- |6||potB59-pomA for PET ||1000||892||100||8.92||8.40807 |- |7||Gad E – for PET||500||1291||100||12.91||5.80945 |- |8||TlpB for PET||1000||901||100||9.01||8.32408 |- |9||DAMP-Pex for PET/pcolA||1000||2089||100||20.89||3.59023 |- |10||Tev Protease for PET||1000||1948||100||19.48||3.8501 |- |11||miRNA switch- miR373- BS for pTET||500||2212||100||22.12||3.3906 |- |12||LacO- DsRed- miR26a-375 pC||1000||1709||100||17.09||4.38853 |- |13||mLacI-miR373 BS for pTRE||1000||1280||100||12.8||5.85938 |- |14||miRNA switch- miR 21 BS- miR 223||1000||1902||100||19.02||3.94322 |- |15||mLacI-miR223 BS miR 21 BS for pTRE||1000||1272||100||12.72||5.89623 |- |16||Trigger RNA for pSB1C3||250||1910||100||19.1||3.9267 |- |17||PsicA for pSB1C3||1000||1546||100||15.46||4.86 |- |18||MVF-sicA for ColA||1000||1042||100||10.42||7.20 |} Not: 100ng pSB1C3 (2050 bp) ≈ 75 fmolPCR of “G-Blocks from IDT” (24.07.2015) {| border="1" class="wikitable" !PCR from G-Bloks |- |||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rev||tetR rev||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |3x (pTEToff)||7.5 ul||7.5 ul||1.5 ul||-||1.5 ul||1.5 ul||1.6 ul||36.9 ul||||58.0 ul |- |15x||37.5 ul||37.5 ul||7.5 ul||7.5 ul||-||7.5 ul||3.0 ul||184.5 ul||||285.0 ul |} {| border="1" class="wikitable" !Cycling for 3x |- |||95˚C||95˚C||57˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |} {| border="1" class="wikitable" !Cycling for 15x |- |||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Results: Bands were at the expected section.
Gel Extraction {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||7/24/2015 12:40:09 PM||-0.8||ng/ul||-0.016||-0.020||0.79||0.24||DNA||50.00 |- |2||pSB1C3||biospec||7/24/2015 12:41:56 PM||42.2||ng/ul||0.848||0.430||1.97||2.05||DNA||50.00 |}
Ligation of „G-Blocks from IDT and pSB1C3“ {| border="1" class="wikitable" !Ligation |- |||Insert DNA||Vector (pSB1C3)||T4 DNA Buffer||T4 DNA Ligase||ddH₂O||Total |- |1||4.9 ul||2.5 ul||2.0 ul||1.0 ul||9.8 ul||20.0 ul |- |2||7.2 ul||2.5 ul||2.0 ul||1.0 ul||7.3 ul||20.0 ul |- |3||9.2 ul||2.5 ul||2.0 ul||1.0 ul||5.3 ul||20.0 ul |- |4||4.8 ul||2.5 ul||2.0 ul||1.0 ul||9.7 ul||20.0 ul |- |5||4.8 ul||2.5 ul||2.0 ul||1.0 ul||9.7 ul||20.0 ul |- |6||8.4 ul||2.5 ul||2.0 ul||1.0 ul||6.1 ul||20.0 ul |- |7||5.8 ul||2.5 ul||2.0 ul||1.0 ul||8.7 ul||20.0 ul |- |8||4.9 ul||2.5 ul||2.0 ul||1.0 ul||9.6 ul||20.0 ul |}
RT 1h
Transformation was made.
(The results of psb1c3 gel extraction was lower. So we performed only the first 7 gene ligation.)
{| border="1" class="wikitable" !1!!Toehold for cola |- |2||TnrA-pTnrA-RFP |- |3||ColA-KanR-dTer |- |4||HNS for PET |- |5||HNS-T108I for PET |- |6||potB59-pomA for PET |- |7||Gad E – for PET |- |8||TlpB for cola (NEB1) |}Creating “Plasmid pCAG” {| border="1" class="wikitable" !Digestion |- |||pTRE (1536 ng/ul)||pCAG (promoter) E||pCAG (promoter) Y||EcoRI||XhoI||Cut Smart Buffer||ddH₂O||Total|| |- |1||3.2 ul||-||-||0.5 ul||0.5 ul||2.0 ul||13.8 ul||20.0 ul||37˚C 2h |- |2||-||10 ul||-||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C 2h |- |3||-||-||10 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight |}
Bands were at the expected section. Gel extraction wil be made.
The Final Concentration
pCAG E: 21.5 ng/ul
pCAG Y: 12 ng/ul{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||eb||biospec||7/25/2015 5:03:57 AM||1.4||ng/ul||0.028||0.006||4.60||0.48||DNA||50.00 |- |2||eb||biospec||7/25/2015 5:05:57 AM||0.0||ng/ul||0.000||-0.012||-0.03||0.06||DNA||50.00 |- |3||ptre delta tre ||biospec||7/25/2015 5:07:44 AM||194.0||ng/ul||3.880||2.033||1.91||2.20||DNA||50.00 |}
Colony PCR of “pSB1C3 – GBlocks” {| border="1" class="wikitable" !PCR from “pCAGGS” |- |||PCR MM||VR fwd||VR rev||ddH₂O||Tag ||DNA||Total|||| |- |1x||14.0 ul||1.0 ul||1.0 ul||7.5 ul||0.2 ul||5.0 ul||28.7 ul|||| |- |33x||462.0 ul||33.0 ul||33.0 ul||247.5 ul||6.6 ul||165.0 ul||940.5 ul|||| |- |33x||462.0 ul||33.0 ul||33.0 ul||247.5 ul||6.6 ul||165.0 ul||940.5 ul|||| |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Sonuc: 8-3 ve 4-9 bands were at the expected section. Liquid Culture was made.
Colony PCR of “G-Blocks” (25.07.2015) {| border="1" class="wikitable" !Colony PCR of “2,3,6,8” |- |||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||24.0 ul |- |6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||73.8 ul||||114.0 ul |} {| border="1" class="wikitable" !Colony PCR of “7,10,12,13” |- |||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||24.0 ul |- |6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||73.8 ul||||114.0 ul |} {| border="1" class="wikitable" !Colony PCR of “1,4,9,15” |- |||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul |- |6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||79.8 ul||||120.0 ul |} {| border="1" class="wikitable" !Colony PCR of “5,16” |- |||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul |- |2X||5.0 ul||5.0 ul||1.0 ul||1.0 ul||1.0 ul||0.4 ul||26.6 ul||||40.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||2’||1.5’||5’||35x |}
G-Blocks PCR / Gel Electrophoresis {| border="1" class="wikitable" !PCR |- |||MgCl₂||(NH₄)2SO₄||CMV fwd||TetR rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul |- |6X||15.0 ul||15.0 ul||3.0 ul||3.0 ul||3.0 ul||1.2 ul||79.8 ul||||120.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||2’||1.5’||5’||35x |}
Result: negative
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||7/26/2015 7:04:13 PM||0.8||ng/ul||0.016||-0.014||-1.13||1.36||DNA||50.00 |- |2||blank||biospec||7/26/2015 7:05:51 PM||-0.4||ng/ul||-0.009||-0.017||0.52||0.17||DNA||50.00 |- |3||colony pcr 8-3||biospec||7/26/2015 7:07:15 PM||87.9||ng/ul||1.758||0.908||1.94||2.09||DNA||50.00 |- |4||colony pcr 4-9||biospec||7/26/2015 7:08:09 PM||101.4||ng/ul||2.027||1.089||1.86||1.74||DNA||50.00 |- |5||colony pcr 4-9||biospec||7/26/2015 7:09:06 PM||71.4||ng/ul||1.429||0.749||1.91||1.96||DNA||50.00 |- |6||colony pcr 8-3||biospec||7/26/2015 7:10:03 PM||87.9||ng/ul||1.758||0.910||1.93||2.10||DNA||50.00 |- |7||colony pcr 4-9||biospec||7/26/2015 7:11:01 PM||57.1||ng/ul||1.142||0.596||1.91||1.77||DNA||50.00 |- |8||colony pcr 4-9||biospec||7/26/2015 7:12:18 PM||77.2||ng/ul||1.543||0.820||1.88||1.76||DNA||50.00 |}Colony PCR of “pSB1C3- GBlocks” {| border="1" class="wikitable" !Gradient PCR from pCAGGS |- |||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||ddH₂O||DNA||Total|| |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||12.3 ul||5.0 ul||25.0 ul|| |- |41X||102.5 ul||102.5 ul||41.0 ul||41.0 ul||20.5 ul||504.3 ul||||881.8 ul|| |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Digestion of “pTEToff” {| border="1" class="wikitable" !Digestion |- |||pTEToff (1141 ng/ul)||SalI (Thermo)||HindIII (Thermo)||Fast Digest Buffer||ddH₂O||Total |- |Volume||3.1 ul||0.5 ul||0.5 ul||2.0 ul||13.9 ul||20.0 ul |}
37˚C 1h
Result: Bands were at the expected section. Gel purification was made.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||7/26/2015 4:18:56 PM||0.3||ng/ul||0.005||-0.010||-0.56||-0.38||DNA||50.00 |- |2||Ptetoff SalI hindIII||biospec||7/26/2015 4:20:23 PM||43.3||ng/ul||0.866||0.452||1.92||0.88||DNA||50.00 |}
Creating “Plasmid pCAG” – Continue (27.07.2015) {| border="1" class="wikitable" !Ligation of „pTRE TRE“ and „Digested pCAG“ |- | |- |||pTRE TRE (194 ng/ul)||pCAG E (21.5 ng/ul)||pCAG Y (12.0 ng/ul)||T4 DNA Ligase||Buffer||ddH₂O||Total |- |1||1.0 ul||2.0 ul||-||0.5 ul||2.0 ul||14.5 ul||20.0 ul |- |2||1.0 ul||-||3.6 ul||0.5 ul||2.0 ul||12.9 ul||20.0 ul |}
Result: On the first plate was six colonies. On the second plate was nine colonies.
Colony PCR will be made.Colony PCR of „pCAG (plasmid)“ {| border="1" class="wikitable" !PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |16X||40.0 ul||40.0 ul||16.0 ul||16.0 ul||8.0 ul||3.2 ul||196.8 ul||||320.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||55˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||2’||1.5’||5’||35x |}
Result: negative
Colony PCR of “pSB1C3 – Gblocks” {| border="1" class="wikitable" !PCR |- |||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |57X||143.0 ul||143.0 ul||57.0 ul||57.0 ul||29.0 ul||11.4 ul||701.0 ul||||1141.4 ul |- |57X||143.0 ul||143.0 ul||57.0 ul||57.0 ul||29.0 ul||11.4 ul||701.0 ul||||1141.4 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Only 16-9 is positive.
Colony PCR of “pSB1C3 – Gblocks” {| border="1" class="wikitable" !Colony PCR of “2,3,8,9,11,13,14” |- |||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |66X||165.0 ul||165.0 ul||66.0 ul||66.0 ul||33.0 ul||13.2 ul||811.8 ul||||1650.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Digestion of “HNS, HNS-T108I, potB59-pomA, GadE, TlpB, DAMP-Pex, Tev Protease” (27.07.2015)
Inserts from GBlocks were digested to ligate with PET45.
{| border="1" class="wikitable" !!!4!!5!!6!!7!!8!!9!!10 |- |HNS (4)||10.0 ul||-||-||-||-||-||- |- |HNS-T108I (5)||-||10.0 ul||-||-||-||-||- |- |potB59-pomA (6)||-||-||10.0 ul||-||-||-||- |- |GadE (7)||-||-||-||10.0 ul||-||-||- |- |TlpB (8)||-||-||-||-||10.0 ul||-||- |- |DAMP-Pex (9)||-||-||-||-||-||10.0 ul||- |- |Tev Protease (10)||-||-||-||-||-||-||10.0 ul |- |Cut Smart Buffer||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul |- |₂XhoI||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul |- |BamHI-HF||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul |- |Milli-Q||7.0 ul||7.0 ul||7.0 ul||7.0 ul||7.0 ul||7.0 ul||7.0 ul |}Total: 20 ul
37˚C overnight
Ligation will be made.Ligation of “GBlocks (4,5,6,7,8,10)” and “PET45 (X+B)” {| border="1" class="wikitable" !!!4!!5!!6!!7!!8!!10 |- |Insert DNA||4.75 ul||4.75 ul||4.75 ul||4.75 ul||4.75 ul||4.75 ul |- |Vector (PET45)||2.20 ul||2.20 ul||.,20 ul||2.20 ul||2.20 ul||2.20 ul |- |Buffer||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul |- |T4 DNA Ligase||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul||0.5 ul |- |ddH₂O||10.55 ul||10.55 ul||10.55 ul||10.55 ul||10.55 ul||10.55 ul |- |Total||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul |} Room Temperature 2h
{| border="1" class="wikitable" !!!11!!14 |- |Insert DNA||3.4 ul||3.95 ul |- |Vector (PET45)||2.20 ul||2.20 ul |- |Buffer||2.0 ul||2.0 ul |- |T4 DNA Ligase||0.5 ul||0.5 ul |- |ddH₂O||11.9 ul||11.35 ul |- |Total||20.0 ul||20.0 ul |} Room Temperature 2h
Creating of “pTRE – mLacI miRNA-BS” {| border="1" class="wikitable" !Digestion of “pTRE” and G-Blocks” |- |||pTRE ||EcoRI-HF||BamHI-HF||mLacI-miR 373 BS||mLacI-miR (21-223) BS||Cut Smart||ddH₂O||Total|| |- |1||3.1 ul||0.5 ul||0.5 ul||-||-||2.0 ul||13.9 ul||20.0 ul||37˚C-3h Gel Elect. |- |2||-||0.5 ul||0.5 ul||10.0 ul||-||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation |- |3||-||0.5 ul||0.5 ul||-||10.0 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation |}
Gel purification was made.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||7/27/2015 2:26:05 PM||0.3||ng/ul||0.007||-0.004||-1.71||0.20||DNA||50.00 |- |2||pTRE e+b||biospec||7/27/2015 2:27:08 PM||92.9||ng/ul||1.859||1.015||1.83||1.24||DNA||50.00 |}
Creating of “pTRE – mLacI miRBS” (27.07.2015) {| border="1" class="wikitable" !Ligation of „pTRE” and “mLacI miRBS” |- |||Digested pTRE||D. mLacI miR373 BS||D. mLacI miR(21-223) BS||T4 DNA Ligase||T4 DNA Buffer||ddH₂O||Total |- |1||1.0 ul||5.9 ul||-||2.0 ul||0.5 ul||10.6 ul||20.0 ul |- |2||1.0 ul||-||5.9 ul||2.0 ul||0.5 ul||10.6 ul||20.0 ul |}
Room Temperature 2h
Transformation was made with TOP10.Creating of “pTEToff – miRBS” (27.07.2015) {| border="1" class="wikitable" !Digestion |- |||pTEToff (2.5 ng/ul)||miRNA switch miR373 BS||miRNA switch miR(223-21) BS||SalI (FD)||HindIII (FD)||Fast Digest Buffer||ddH₂O||Total|| |- |1||2.0 ul||-||-||0.5 ul||0.5 ul||5.0 ul||42.0 ul||50.0 ul||37˚C-3h Gel Elect. |- |2||-||10.0 ul||-||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation |- |3||-||-||10.0 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight/80˚C-10’ heat inactivation |}
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||7/27/2015 8:35:05 PM||0.0||ng/ul||0.000||-0.013||-0.01||0.01||DNA||50.00 |- |2||pTRE s+h||biospec||7/27/2015 8:36:16 PM||76.7||ng/ul||1.534||0.810||1.89||1.05||DNA||50.00 |}
Creating of “pTEToff – miRNA Switch miR BS” (28.07.2015) {| border="1" class="wikitable" !Ligation of „pTRE” and “mLacI miRBS” |- |||Digested pTEToff||D. mLacI miR373 BS||D. mLacI miR(21-223) BS||T4 DNA Ligase||T4 DNA Buffer||ddH₂O||Total |- |1||2.2 ul||3.4 ul||-||2.0 ul||0.5 ul||10.9 ul||20.0 ul |- |2||2.2 ul||-||4.0 ul||2.0 ul||0.5 ul||11.3 ul||20.0 ul |}
Room Tempretare 2h
Transformation was made.Creating of “pCAG” {| border="1" class="wikitable" !Digestion of “pCAG (Promotor)” |- |||pCAG (promoter) E||pCAG (promoter) Y||EcoRI-HF||XhoI-HF||Cut Smart Buffer||ddH₂O||Total|||| |- |1||10.0 ul||-||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight|| |- |2||-||10.0 ul||0.5 ul||0.5 ul||2.0 ul||7.0 ul||20.0 ul||37˚C overnight|| |}
Mini Prep of “Colony PCR 16-9” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||7/28/2015 12:11:17 PM||-1.9||ng/ul||-0.038||-0.049||0.77||0.19||DNA||50.00 |- |2||blank||biospec||7/28/2015 12:12:22 PM||-0.4||ng/ul||-0.007||-0.014||0.53||0.30||DNA||50.00 |- |3||colony pcr 16-9||biospec||7/28/2015 12:13:33 PM||84.9||ng/ul||1.698||0.881||1.93||2.02||DNA||50.00 |- |4||colony pcr 16-9||biospec||7/28/2015 12:14:24 PM||76.3||ng/ul||1.527||0.797||1.92||1.95||DNA||50.00 |}
Mini Prep of “pTEToff” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||7/30/2015 4:44:01 PM||0.4||ng/ul||0.008||-0.005||-1.55||1.11||DNA||50.00 |- |2||ptetoff neb||biospec||7/30/2015 4:45:00 PM||442.2||ng/ul||8.884||4.683||1.89||2.20||DNA||50.00 |- |3||ptetoff bl21||biospec||7/30/2015 4:46:08 PM||314.3||ng/ul||6.286||3.317||1.90||2.22||DNA||50.00 |}
Cut Check of “HNS toehold and Trigger RNA” (30.07.2015) {| border="1" class="wikitable" !!!HNS (70 ng/ul)!!TlpB (87 ng/ul)!!Trigger RNA (80 ng/ul)!!EcoRI (Thermo)!!PstI (Thermo)!!Fast Digest Buffer!!ddH₂O!!Total!! |- |1||3.5 ul||-||-||1.0 ul||1.0 ul||2.0 ul||12.5 ul||20.0 ul||37˚C 20min |- |2||-||3.0 ul||-||1.0 ul||1.0 ul||2.0 ul||13.0 ul||20.0 ul||37˚C 20min |- |3||-||-||3.0 ul||1.0 ul||1.0 ul||2.0 ul||13.0 ul||20.0 ul||37˚C 20min |}
Gel Electropheres was made.
Result: Bands were at the expected section.
HNS: 480 bp
tgRNA:˞ 100bp
Toehold: ˃1000bp
Colony PCR of „9-10 GBlocks“
Result: negative
Expected: ˞1100 bp
Observed: ˞300 bp
Gel extraction 1-6 {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/2/2015 12:01:41 AM||0.2||ng/ul||0.004||-0.002||-2.02||17.14||DNA||50.00 |- |2||psb1c3||biospec||8/2/2015 12:31:14 AM||43.0||ng/ul||0.861||0.462||1.87||0.94||DNA||50.00 |}
Gel extraction 8-9 {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/2/2015 12:27:36 AM||-0.4||ng/ul||-0.009||-0.012||0.74||-0.09||DNA||50.00 |- |2||psb1c3 atoms||biospec||8/2/2015 12:34:11 AM||10.4||ng/ul||0.208||0.111||1.87||0.03||DNA||50.00 |}
Colony PCR of „pSB1C3 – Gblocks“ {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul |- |17X||42.5 ul||42.5 ul||8.5 ul||8.5 ul||8.5 ul||3.4 ul||226.1 ul||||340.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
August
11.08. We started designing our wiki.
18.08.2015 We got a little tired of doing experiments, so treated ourselves with an awesome team dinner at a fish restaurant.
22.08.2015 A team dinner again, but this time our dear professors Mehmet and Esra Gündüz hosted us at their house.
29.08.2015 Wiki’s tour part is drawn, now it’s time to color these drawings!
Ligation of „pSB1C3 – Gblocks“ {| border="1" class="wikitable" !!!2!!3!!5!!6!!7!!8!!9!!10!!11!!12!!13!!14!!15!! |- |Insert||7.3 ul||7.0 ul||4.8 ul||8.4 ul||5.8 ul||8.3 ul||3.6 ul||3.9 ul||3.4 ul||4.4 ul||5.9 ul||3.9 ul||5.9 ul|| |- |Vector||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul|| |- |Buffer||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul||2.0 ul|| |- |Enzyme||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul||1.0 ul|| |- |ddH₂O||7.7 ul||8.0 ul||10.2 ul||6.6 ul||9.2 ul||6.7 ul||11.4 ul||11.1 ul||11.6 ul||10.6 ul||9.1 ul||11.1 ul||9.1 ul|| |- |Total||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul||20.0 ul|| |}
Result: negative
Colony PCR of „pSB1C3 – Gblocks #14“ {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||VR fwd||VR rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |36X||90.0 ul||90.0 ul||36.0 ul||36.0 ul||18.0 ul||7.2 ul||442.8 ul||||720.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Digestion of “pSB1C3-RFP” {| border="1" class="wikitable" !Digestion of “pSB1C3-RFP” |- |||Vector||Fast Digest Buffer||EcoRI (FD)||PstI (FD)||ddH₂O||Total |- |1x||2.0 ul||2.0 ul||1.0 ul||1.0 ul||14.0 ul||20.0 ul |}
37˚C 2h
Gel Electropheresis was made.
Result: Bands were at the expected section. Gel purification was made.
Ligation of “pSB1C3 – Gblock #14” {| border="1" class="wikitable" !Ligation of “pSB1C3 – Gblock #14” |- |||Insert||Vector||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total |- |1) PCR clean up vektörü ile 2.5 ul||2.5 ul||2.0 ul |- |(100ng)||2.0 ul |- |||1.0 ul||12.5 ul||20.0 ul |- |2) Gel Extraction 7.5 ul||7.5 ul||0.8 ul |- |(50ng)||2.0 ul |- |||1.0 ul||8.7 ul||20.0 ul |}
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/3/2015 6:10:02 PM||0.2||ng/ul||0.004||-0.010||-0.41||1.13||DNA||50.00 |- |2||psb1c3 gel||biospec||8/3/2015 6:12:16 PM||62.5||ng/ul||1.250||0.664||1.88||0.90||DNA||50.00 |- |4||psb1c3 clean-up||biospec||8/3/2015 6:13:39 PM||170.5||ng/ul||3.409||1.884||1.81||1.33||DNA||50.00 |}
PCR of “G-Bloks via Q5 Polymerase” (04.08.2015) {| border="1" class="wikitable" !PCR!!!!!!!!!!!! |- |||SV40 poly A rev||CMV fwd||tetR rev||2x Q5 Master Mix||DNA||Total |- |1x||2.5 ul||2.5 ul||2.5 ul||10.0 ul||5.0 ul||20.0 ul |}
{| border="1" class="wikitable" !Cycling SV40 rev/CMV fwd |- |||95˚C||95˚C||63˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1’||2’||35x |}
{| border="1" class="wikitable" !Cycling CMV fwd/tetR rev |- |||98˚C||98˚C||66˚C||72˚C||72˚C||Cycle |- |Time||30’’||10’’||30’’||1’||2’||35x |}
Digestion of “pSB1C3-RFP” {| border="1" class="wikitable" !Digestion!!!!!!!!!!!! |- |||Vector||Cut Smart Buffer||EcoRI||SpeI||ddH₂O||Total |- |1x||2.0 ul||2.0 ul||1.0 ul||1.0 ul||14.0 ul||20.0 ul |}
37˚C 4h
Result: Bands were at the expected section.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/4/2015 7:03:09 AM||0.9||ng/ul||0.017||0.005||3.41||0.68||DNA||50.00 |- |2||Blank2||biospec||8/4/2015 7:04:51 AM||-0.5||ng/ul||-0.010||-0.017||0.59||0.45||DNA||50.00 |- |4||clean up||biospec||8/4/2015 7:05:55 AM||86.8||ng/ul||1.735||0.913||1.90||0.40||DNA||50.00 |- |5||gel ext ||biospec||8/4/2015 7:06:51 AM||36.2||ng/ul||0.723||0.380||1.90||0.29||DNA||50.00 |}
Colony PCR of “11,12,13 pSB1C3 - GBlocks” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||VF||VR||Tag||ddH₂O||DNA||Total|| |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul|| |- |17X||42.5 ul||42.5 ul||8.5 ul||8.5 ul||3.4 ul||226.1 ul||||340.0 ul|| |} {| border="1" class="wikitable" !Cycling CMV fwd/tetR rev |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Ligation and Transformation of “5,6,7,8,9 pSB1C3-GBlocks” {| border="1" class="wikitable" !Ligation of “GBlocks 11-12 pSB1C3” |- |||Vector||Gen||Buffer||Ligase||ddH₂O||Total |- |5||0.8 ul||2.37 ul||1.0 ul||0.5 ul||5.33 ul||10.0 ul |- |6||0.8 ul||4.2 ul||1.0 ul||0.5 ul||3.5 ul||10.0 ul |- |7||0.8 ul||2.94 ul||1.0 ul||0.5 ul||4.76 ul||10.0 ul |- |8||0.8 ul||4.16 ul||1.0 ul||0.5 ul||3.54 ul||10.0 ul |- |9||0.8 ul||1.79 ul||1.0 ul||0.5 ul||5.91 ul||10.0 ul |}
50ng DNA
1 vector:0.5 insert
Transfomation with TOP10.
Ligation of “GBlocks 11-12 pSB1C3” {| border="1" class="wikitable" !Ligation of “GBlocks 11-12 pSB1C3” |- |||pSB1C3||Insert||Buffer||Ligase||ddH₂O||Total |- |11||0.8 ul||1.7 ul||1.0 ul||0.5 ul||6.0 ul||10.0 ul |- |12||0.4 ul||1.1 ul||1.0 ul||0.5 ul||7.0 ul||10.0 ul |} Transformation was made.
Colony PCR of “5,6,7,8,9,T7 system – Gblocks” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.1 ul||13.3 ul||5.0 ul||24.9 ul |- |58X||145.0 ul||145.0 ul||29.0 ul||29.0 ul||29.0 ul||5.8 ul||771.4 ul||||1154.2 ul |} {| border="1" class="sortable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Ligation of “GBlocks 2,5,6,7,8,9,10,12,13,15,17 pSB1C3-RFP” {| border="1" class="wikitable" !Ligation of “GBlocks 11-12 pSB1C3” |- |||pSB1C3||Insert||Buffer||Ligase||ddH₂O||Total |- |2||1.4 ul||3.65 ul||1.0 ul||0.5 ul||3.45 ul||10.0 ul |- |5||1.4 ul||2.4 ul||1.0 ul||0.5 ul||4.7 ul||10.0 ul |- |6||1.4 ul||4.2 ul||1.0 ul||0.5 ul||2.9 ul||10.0 ul |- |7||1.4 ul||2.9 ul||1.0 ul||0.5 ul||4.2 ul||10.0 ul |- |8||1.4 ul||4.15 ul||1.0 ul||0.5 ul||2.95 ul||10.0 ul |- |9||1.4 ul||1.8 ul||1.0 ul||0.5 ul||5.3 ul||10.0 ul |- |10||1.4 ul||1.9 ul||1.0 ul||0.5 ul||5.2 ul||10.0 ul |- |12||1.4 ul||2.2 ul||1.0 ul||0.5 ul||4.9 ul||10.0 ul |- |13||1.4 ul||2.9 ul||1.0 ul||0.5 ul||4.2 ul||10.0 ul |- |15||1.4 ul||2.95 ul||1.0 ul||0.5 ul||4.15 ul||10.0 ul |- |17||1.4 ul||1.25 ul||1.0 ul||0.5 ul||5.75 ul||10.0 ul |} Transformation was made.
Colony PCR of “pTRE-1,13,15” (06.08.2015) {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||CMV fwd||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |20X||50.0 ul||50.0 ul||20.0 ul||20.0 ul||10.0 ul||2.0 ul||246.0 ul||||400.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
GEL GÖRÜNTÜSÜColony PCR of “5,6,7,9 – Gblocks” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||0.5 ul||0.5 ul||0.5 ul||0.2 ul||13.3 ul||5.0 ul||25.0 ul |- |40X||100.0 ul||100.0 ul||20.0 ul||20.0 ul||20.0 ul||8.0 ul||532.0 ul||||800.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Colony PCR of “7,8,10 – Gblocks” from Masterplate {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||T7 terminatör Rev.||T7 terminatör For.||dNTP||Tag||ddH₂O||DNA||Totalzz |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.5 ul||12.3 ul||5.0 ul||25.0 ul |- |26X||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||13.0 ul||319.8 ul||||527.8 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Digestion of “pET45” {| border="1" class="wikitable" !Digestion |- |||pET45 (455ng/ul)||pSB1C3-RFP (400 ng/ul)||Cut Smart Buffer||XhoI (Neb)||BamHI-HF (Neb)||EcoRI-HF (Neb)||SpeI (Neb)||ddH₂O||Total |- |1||-||5.0 ul||2.0 ul||-||-||1.0 ul||1.0 ul||11.0 ul||20.0 ul |- |2||4.0 ul||-||2.0 ul||1.0 ul||1.0 ul||-||-||12.0 ul||20.0 ul |}
37˚C 3.5h
Result: Positive. Gel extraction was made.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/9/2015 5:04:14 PM||-0.6||ng/ul||-0.011||0.005||3.41||0.68||DNA||50.00 |- |2||pet45 x+b||biospec||8/9/2015 5:05:26 PM||106.9||ng/ul||2.138||-0.017||0.59||0.45||DNA||50.00 |- |4||psb1c3 e+s||biospec||8/9/2015 5:06:20 PM||133.3||ng/ul||2.666||0.913||1.90||0.40||DNA||50.00 |} Ligation of
“PET45 – HNS” {| border="1" class="wikitable"
!Ligation |- |||Vector||Insert||T4 DNA Ligase
Buffer||T4 DNA Ligase||ddH₂O||Total |-
|1x||2.1 ul||1.0 ul||2.0 ul||0.5 ul||14.4
ul||20.0 ul |}
16˚C 2h
Digestion of “pSB1C3 – HNS” {| border="1" class="wikitable" !Ligation |- |||Gen||BamHI||XhoI||Cut Smart Buffer||ddH₂O||Total |- |1x||11.5 ul||1.0 ul||1.0 ul||2.0 ul||4.5 ul||20.0 ul |} 37˚C 3h
Colony PCR of “pTRE #13” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||CMV for.||SV40 rev.||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |10X||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||250.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||60˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Colony PCR of “PET45-4,5,6,7,8,9,10” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||T7 ter. fw||T7 ter. rev||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |86X||215.0 ul||215.0 ul||86.0 ul||186.0 ul||43.0 ul||17.2 ul||1075.0 ul||||1720.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Bands were at the expected section. Liquid Culture was made.
Ligation of “pColA-KanR and pSB1C3-Toehold” {| border="1" class="wikitable" !Ligation!! |- |||pColA-KanR (500 ng/ul)||pSb1C3-Toehold||T4 DNA Ligase Buffer (Thermo)||T4 DNA Ligase (Thermo)||ddH₂O||Total||Vector:Insert Ratios |- |1||2.0 ul||1.0 ul||2.0 ul||1.0 ul||14.0 ul||20.0 ul||1:0.33 |- |2||2.0 ul||3.0 ul||2.0 ul||1.0 ul||12.0 ul||20.0 ul||1:1 |- |-K||2.0 ul||-||2.0 ul||1.0 ul||15.0 ul||20.0 ul||- |}
22˚C 1h
Mini Prep of “pColA-3,4,5” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/11/2015 11:11:42 PM||-0.2||ng/ul||-0.003||-0.007||0.49||0.13||DNA||50.00 |- |2||ColA-3||biospec||8/11/2015 11:13:07 PM||27.2||ng/ul||0.544||0.260||2.10||1.95||DNA||50.00 |- |3||ColA-4||biospec||8/11/2015 11:14:38 PM||25.5||ng/ul||0.509||0.253||2.01||1.82||DNA||50.00 |- |4||ColA-4(2)||biospec||8/11/2015 11:15:58 PM||25.8||ng/ul||0.517||0.255||2.022||1.81||DNA||50.00 |- |5||ColA-5||biospec||8/11/2015 11:17:02 PM||44.4||ng/ul||0.887||0.449||1.98||1.76||DNA||50.00 |}
Result: The Band of pColA were at the expected section. 2000 pb
Ligation of “#2 GBlock with pET45” (11.08.2015) {| border="1" class="wikitable" !Ligation |- |||pET45 (100 ng/ul)||#2 GBlock||T4 DNA Ligase Buffer ||T4 DNA Ligase ||ddH₂O||Total |- |1x||1.0 ul||7.3 ul||2.0 ul||1.0 ul||8.7 ul||20.0 ul |}
22˚C - 1’40’’
Transformation was made.Mini Prep {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/12/2015 7:38:28 PM||0.4||ng/ul||0.008||-0.001||-8.91||0.76||DNA||50.00 |- |2||pet45 4-4||biospec||8/12/2015 7:40:21 PM||40.3||ng/ul||0.807||0.402||2.01||2.44||DNA||50.00 |- |3||pet45 4-3||biospec||8/12/2015 7:41:10 PM||53.1||ng/ul||1.063||0.570||1.86||1.74||DNA||50.00 |- |4||pet45 8-12||biospec||8/12/2015 7:41:55 PM||61.8||ng/ul||1.236||0.642||1.92||2.08||DNA||50.00 |- |5||pet45 8-3||biospec||8/12/2015 7:42:37 PM||32.9||ng/ul||0.658||0.332||1.98||2.00||DNA||50.00 |- |6||pet45 9-11||biospec||8/12/2015 7:44:03 PM||34.5||ng/ul||0.690||0.345||2.00||1.77||DNA||50.00 |- |7||pet45 8-1||biospec||8/12/2015 7:44:54 PM||39.0||ng/ul||0.780||0.408||1.91||1.72||DNA||50.00 |- |8||pet45 5-10||biospec||8/12/2015 7:45:49 PM||43.2||ng/ul||0.865||0.441||1.96||1.81||DNA||50.00 |- |9||pet45 6-3||biospec||8/12/2015 7:46:42 PM||43.5||ng/ul||0.870||0.437||1.99||1.92||DNA||50.00 |- |10||pet45 9-8||biospec||8/12/2015 7:47:23 PM||48.1||ng/ul||0.961||0.496||1.94||1.88||DNA||50.00 |- |11||pet45 7-4||biospec||8/12/2015 7:48:06 PM||36.2||ng/ul||0.723||0.372||1.94||1.97||DNA||50.00 |- |12||pet45 4-1||biospec||8/12/2015 7:48:50 PM||49.4||ng/ul||0.987||0.509||1.94||1.99||DNA||50.00 |- |13||pet45 4-2||biospec||8/12/2015 7:49:43 PM||59.3||ng/ul||1.187||0.606||1.96||2.09||DNA||50.00 |- |14||pet45 7-3||biospec||8/12/2015 7:50:33 PM||30.2||ng/ul||0.604||0.307||1.97||1.96||DNA||50.00 |- |15||psb1c3 12||biospec||8/12/2015 7:51:18 PM||141.1||ng/ul||2.823||1.462||1.93||2.14||DNA||50.00 |- |16||pet45 5-7||biospec||8/12/2015 7:52:04 PM||26.1||ng/ul||0.522||0.265||1.97||2.00||DNA||50.00 |- |17||pet45 5-3||biospec||8/12/2015 7:52:49 PM||46.1||ng/ul||0.923||0.470||1.96||2.02||DNA||50.00 |- |18||pet45 6-12||biospec||8/12/2015 7:53:36 PM||29.3||ng/ul||0.586||0.283||2.07||1.80||DNA||50.00 |- |19||pet45 1-12||biospec||8/12/2015 7:54:25 PM||21.4||ng/ul||0.428||0.210||2.04||1.72||DNA||50.00 |- |20||pet45 5-8||biospec||8/12/2015 7:55:08 PM||29.1||ng/ul||0.582||0.299||1.95||2.07||DNA||50.00 |- |21||psb1c3 1||biospec||8/12/2015 7:55:51 PM||75.3||ng/ul||1.507||0.779||1.93||2.07||DNA||50.00 |- |22||pet45 8-4||biospec||8/12/2015 7:56:33 PM||44.1||ng/ul||0.882||0.451||1.96||1.99||DNA||50.00 |- |23||pet45 7-2||biospec||8/12/2015 7:57:16 PM||25.3||ng/ul||0.506||0.259||1.95||1.70||DNA||50.00 |}
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/12/2015 7:17:01 PM||-0.3||ng/ul||-0.007||-0.015||0.47||0.23||DNA||50.00 |- |2||psb1c3-4hns||biospec||8/12/2015 7:18:16 PM||61.9||ng/ul||1.237||0.652||1.90||2.17||DNA||50.00 |- |3||pet45 10-1||biospec||8/12/2015 7:19:32 PM||46.7||ng/ul||0.934||0.486||1.92||2.00||DNA||50.00 |- |4||pet45 10-6||biospec||8/12/2015 7:20:23 PM||52.0||ng/ul||1.040||0.549||1.89||1.88||DNA||50.00 |- |5||pet45 10-3||biospec||8/12/2015 7:21:14 PM||51.9||ng/ul||1.037||0.536||1.94||2.10||DNA||50.00 |- |6||pet45 9-6||biospec||8/12/2015 7:22:10 PM||37.7||ng/ul||0.755||0.389||1.94||1.99||DNA||50.00 |- |7||pet45 10-8||biospec||8/12/2015 7:23:08 PM||85.3||ng/ul||1.705||1.002||1.70||0.93||DNA||50.00 |- |8||psb1c3 16-trigger rna||biospec||8/12/2015 7:24:27 PM||125.7||ng/ul||2.514||1.401||1.79||1.32||DNA||50.00 |- |9||pet45 10||biospec||8/12/2015 7:25:23 PM||84.7||ng/ul||1.694||1.025||1.65||0.76||DNA||50.00 |- |10||pet45 6-6||biospec||8/12/2015 7:26:23 PM||100.3||ng/ul||2.006||1.124||1.79||1.24||DNA||50.00 |- |11||pet45 8-2||biospec||8/12/2015 7:27:20 PM||67.5||ng/ul||1.349||0.778||1.73||0.91||DNA||50.00 |- |12||pet45 7-1||biospec||8/12/2015 7:28:07 PM||52.5||ng/ul||1.050||0.549||1.91||1.83||DNA||50.00 |}
Cut Check of “pET45 1,4,5,6,7,8,9,10 – BamHI/XhoI” {| border="1" class="wikitable" !Ligation |- |||Gen (250 ng/ul)||Cut Smart Buffer||BamHI (NEB)||XhoI (NEB)||ddH₂O||Total |- |1||11.7 ul||2.0 ul||1.0 ul||1.0 ul||4.3 ul||20.0 ul |- |4||6.2 ul||2.0 ul||1.0 ul||1.0 ul||9.8 ul||20.0 ul |- |5||8.6 ul||2.0 ul||1.0 ul||1.0 ul||7.4 ul||20.0 ul |- |6||8.6 ul||2.0 ul||1.0 ul||1.0 ul||7.4 ul||20.0 ul |- |7||8.3 ul||2.0 ul||1.0 ul||1.0 ul||7.7 ul||20.0 ul |- |8||7.4 ul||2.0 ul||1.0 ul||1.0 ul||8.6 ul||20.0 ul |- |9||6.7 ul||2.0 ul||1.0 ul||1.0 ul||9.3 ul||20.0 ul |- |10||5.9 ul||2.0 ul||1.0 ul||1.0 ul||10.1 ul||20.0 ul |}
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/13/2015 1:31:59 AM||0.0||ng/ul||-0.001||-0.005||0.17||0.08||DNA||50.00 |- |2||psb1c3-e+p||biospec||8/13/2015 1:32:58 AM||58.6||ng/ul||1.172||0.618||1.90||0.57||DNA||50.00 |}
Colony PCR of “pTRE – 12,13 GBlocks” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||CMV fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |15X||37.5 ul||37.5 ul||15.0 ul||15.0 ul||7.5 ul||3.0 ul||184.5 ul||||300.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Colony PCR of “pColA – Toehold GFP” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |26X||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||5.2 ul||319.8 ul||||520.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: 2-4, 2-8, 1-6 Bands were at the expected section.
Digestion of “pSB1C3-RFP” {| border="1" class="wikitable" !Digestion |- |||pSB1C3-RFP (400 ng/ul)||Cut Smart Buffer ||EcoRI-HF||PstI||ddH₂O||Total |- |1||5.0 ul||2.0 ul||1.0 ul||1.0 ul||11.0 ul||20.0 ul |- |2||5.0 ul||2.0 ul||1.0 ul||1.0 ul||11.0 ul||20.0 ul |- |3||5.0 ul||2.0 ul||1.0 ul||1.0 ul||11.0 ul||20.0 ul |}
37˚C – 3.5h
Gel extraction was made.Ligation of “pSB1C3-GBlocks” {| border="1" class="wikitable" !Ligation |- |||Digested pSB1C3||GBlocks ||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total |- |1||1.7 ul||2.0 ul||2.0 ul||1.0 ul||13.3 ul||20.0 ul |}
22˚C – 1.30h
Transformation with Neb10-β. Colony PCR will be made.Colony PCR of “GBlocks – 2,5,6,7,8,9” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |45X||112.5 ul||112.5 ul||45.0 ul||45.0 ul||22.5 ul||9.0 ul||553.5 ul||||900.0 ul |- |55X||137.5 ul||137.5 ul||55.0 ul||55.0 ul||27.5 ul||11.0 ul||676.5 ul||||1100.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Digestion/Gel Electrophoresis/Gel Extraction and Purification of “pSB1C3” {| border="1" class="wikitable" !Digestion |- |||DNA||FD||EcoRI||PstI||ddH₂O||Total |- |1x||2.5 ul||2.0 ul||1.0 ul||1.0 ul||13.5 ul||20.0 ul |}
37˚C 1h
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/16/2015 10:45:30 PM||0.1||ng/ul||0.003||-0.017||0.17||-0.14||DNA||50.00 |- |2||psb1c3-e+p||biospec||8/16/2015 10:47:18 PM||14.0||ng/ul||0.279||0.145||1.92||0.15||DNA||50.00 |}
Colony PCR of “pSB1C3 7,10,12,13 and pET45 2” {| border="1" class="wikitable" !Colony PCR of “pSB1C3 7,10,12,13” |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |6X||15.0 ul||15.0 ul||6.0 ul||6.0 ul||3.0 ul||1.2 ul||73.8 ul||||120.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/16/2015 8:54:05 PM||-0.2||ng/ul||-0.004||-0.017||0.25||0.12||DNA||50.00 |- |2||psb1c3 13-15||biospec||8/16/2015 8:55:29 PM||125.2||ng/ul||2.505||1.339||1.87||2.01||DNA||50.00 |- |3||psb1c3 15-19||biospec||8/16/2015 8:56:24 PM||93.5||ng/ul||1.871||0.999||1.87||1.94||DNA||50.00 |- |4||psb1c3 7-16||biospec||8/16/2015 8:57:33 PM||149.4||ng/ul||2.988||1.575||1.90||2.14||DNA||50.00 |- |5||psb1c3 10-16||biospec||8/16/2015 8:58:18 PM||138.0||ng/ul||2.760||1.469||1.88||2.13||DNA||50.00 |- |6||psb1c3 10-17||biospec||8/16/2015 8:59:03 PM||185.5||ng/ul||3.710||1.994||1.86||1.86||DNA||50.00 |- |7||psb1c3 7-11||biospec||8/16/2015 8:00:54 PM||73.8||ng/ul||1.475||0.843||1.75||1.08||DNA||50.00 |- |8||psb1c3 13-14||biospec||8/16/2015 8:01:53 PM||178.7||ng/ul||3.575||1.922||1.86||1.87||DNA||50.00 |- |9||psb1c3 7-20||biospec||8/16/2015 8:02:54 PM||73.6||ng/ul||1.472||0.774||1.90||2.05||DNA||50.00 |- |10||pcola toe 2-4||biospec||8/16/2015 8:03:57 PM||40.1||ng/ul||0.801||0.441||1.82||1.13||DNA||50.00 |- |11||pcola toe 2-8||biospec||8/16/2015 8:05:31 PM||48.7||ng/ul||0.974||0.503||1.94||1.61||DNA||50.00 |- |12||pcola toe 1-6||biospec||8/16/2015 8:06:09 PM||54.9||ng/ul||1.098||0.570||1.93||1.62||DNA||50.00 |- |13||pcola blunt 1||biospec||8/16/2015 8:06:52 PM||23.9||ng/ul||0.479||0.244||1.96||1.24||DNA||50.00 |- |14||pcola blunt 6||biospec||8/16/2015 8:07:30 PM||31.1||ng/ul||0.623||0.314||1.98||1.67||DNA||50.00 |}
Ligation of “pColA
and pSB1C3 #9 (Damp-Pex)” {| border="1"
class="wikitable" !Ligation |- |||pColA (Vector
50 ng/ul)||Insert (Damp-Pex)||T4 DNA Ligase
Buffer||T4 DNA
Ligase||ddH₂O||Total||Vector:Insert Ratio |-
|9.1||2.0 ul||1.0 ul||2.0 ul||1.0 ul||14.0
ul||20.0 ul||1:0.33 |- |9.2||2.0 ul||2.8
ul||2.0 ul||1.0 ul||12.2 ul||20.0 ul||1:1
|}Miniprep of “pTet-off DH5α /Top10/NEB5fα/NEB10β” /{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/18/2015 3:04:27 PM||0.4||ng/ul||0.008||-0.008||-1.01||1.01||DNA||50.00 |- |2||top10||biospec||8/18/2015 3:05:19 PM||258.5||ng/ul||5.169||2.753||1.88||2.11||DNA||50.00 |- |3||neb5a||biospec||8/18/2015 3:06:25 PM||30.6||ng/ul||0.612||0.324||1.89||1.34||DNA||50.00 |- |4||neb5a||biospec||8/18/2015 3:07:30 PM||33.4||ng/ul||0.668||0.350||1.91||1.27||DNA||50.00 |- |5||neb10beta||biospec||8/18/2015 3:08:22 PM||214.6||ng/ul||4.292||2.264||1.90||2.19||DNA||50.00 |}
Colony PCR of “pET45 #2.8” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |25X||62.5 ul||62.5 ul||25.0 ul||25.0 ul||12.5 ul||5.0 ul||307.5 ul||||500.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Colony PCR of “pSB1C3 17,6,8” {| border="1" class="wikitable" !Colony PCR of “pSB1C3 17,6,8” |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |32X||80.0 ul||80.0 ul||32.0 ul||32.0 ul||16.0 ul||6.4 ul||393.6 ul||||640.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: 6: Liquid Culture was made.
8: sequencing will be done
17: Colony PCR will be repeated.
Colony PCR of “pColA blunt” {| border="1" class="wikitable" !Colony PCR of “pColA blunt” |- |||MgCl₂||(NH₄)2SO₄||ColA fw||ColA rev||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |20X||50.0 ul||50.0 ul||20.0 ul||20.0 ul||10.0 ul||4.0 ul||246.0 ul||||400.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
PCR of “Q5 DNA Polymerase for pCAG” {| border="1" class="wikitable" !PCR |- |||Template DNA||pCAG fw||pCAG rev||Master 2x Mix||ddH₂O||Total |- |1x||1.0 ul||5.0 ul||5.0 ul||25.0 ul||14.0 ul||50.0 ul |} {| border="1" class="wikitable" !Cycling |- |||98˚C||98˚C||70˚C||72˚C||72˚C||Cycle |- |Time||30’’||10’’||30’’||1’||5’||35x |}
Result: Bands were at the expected section. PCR clean up will be made.
Digestion of “pCAG – Clean up” {| border="1" class="wikitable" !Digestion |- |||pCAG – Clean up (34 ng/ul)||pCAG – Clean up (61.6 ng/ul)||EcoRI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total |- |1||7.4 ul||-||0.5 ul||0.5 ul||2.0 ul||9.6 ul||20.0 ul |- |2||-||4.1 ul||0.5 ul||0.5 ul||2.0 ul||12.9 ul||20.0 ul |}
37˚C overnight
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/19/2015 7:08:39 PM||0.0||ng/ul||0.000||0.000||27.59||-0.07||DNA||50.00 |- |2||pcag||biospec||8/19/2015 7:10:01 PM||61.6||ng/ul||1.233||0.686||1.80||1.02||DNA||50.00 |}
Digestion of “pSB1C3 – mLacI miR373-BS” {| border="1" class="wikitable" !Digestion |- |||mLacI miR373-BS||EcoRI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total|| |- |1||3.4 ul||0.5 ul||0.5 ul||2.0 ul||13.9 ul||20.0 ul|| |}
37˚C 2.30h
Ligation of “pSB1C3 – mLacI miR373-BS with pTRE” {| border="1" class="wikitable" !Ligation |- |||Digested mLacI miR373-BS with pTRE||pTRE||T4 DNA Ligase Buffer||T4 DNA Ligase||Cut Smart Buffer||ddH₂O||Total |- |1||3.0 ul||1.0 ul||2.0 ul||1.0 ul||2.0 ul||13.0 ul||20.0 ul |}
22˚C 1.30h
Transformation will be made.
Colony PCR of “#6,8” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||ColA fw||ColA rev||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |25X||62.5 ul||62.5 ul||25.0 ul||25.0 ul||12.5 ul||5.0 ul||307.5 ul||||500.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Positive. Liquid Culture was made.
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/22/2015 12:11:34 PM||0.5||ng/ul||0.010||-0.003||-3.39||-3.90||DNA||50.00 |- |2||8-1||biospec||8/22/2015 12:12:38 PM||70.3||ng/ul||1.406||0.755||1.86||1.64||DNA||50.00 |- |3||8-3||biospec||8/22/2015 12:13:38 PM||80.7||ng/ul||1.613||0.846||1.91||2.11||DNA||50.00 |- |4||6-1||biospec||8/22/2015 12:14:32 PM||33.3||ng/ul||0.666||0.354||1.88||1.44||DNA||50.00 |}
Cut Check of “#6,8” {| border="1" class="wikitable" !Cut Check |- |||DNA (400 ng/ul)||EcoRI||PstI||Fast Digest Buffer||ddH₂O||Total |- |1||5.7 ul||0.5 ul||0.5 ul||2.0 ul||11.3 ul||20.0 ul |- |2||4.8 ul||0.5 ul||0.5 ul||2.0 ul||12.2 ul||20.0 ul |- |3||12.0 ul||0.5 ul||0.5 ul||2.0 ul||5.0 ul||20.0 ul |}
37˚C 40’
Result: positive
Colony PCR “pSB1C3 17,6,8 – pColA, Damp-Pex, pTetoff”
Mini Prep of “pSB1C3-#17” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/20/2015 5:04:31 PM||0.6||ng/ul||0.013||0.007||1.93||1.40||DNA||50.00 |- |2||psb1c3 17-2||biospec||8/20/2015 5:05:28 PM||78.9||ng/ul||1.579||0.821||1.92||2.17||DNA||50.00 |- |3||psb1c3 17-5||biospec||8/20/2015 5:06:18 PM||77.5||ng/ul||1.551||0.806||1.92||2.08||DNA||50.00 |- |4||psb1c3 17-8||biospec||8/20/2015 5:07:02 PM||87.5||ng/ul||1.749||0.910||1.92||2.08||DNA||50.00 |- |5||psb1c3 17-10||biospec||8/20/2015 5:07:47 PM||105.4||ng/ul||2.108||1.087||1.94||2.15||DNA||50.00 |}
Digestion of “pSB1C3 T7-10 and pET45 #4-1, #5-3, #6-6, #8-15” (21.08.2015) {| border="1" class="wikitable" !Digestion pET45 #4-1, #5-3, #6-6, #8-15 |- |||DNA||BamHI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total|| |- |#4-1||10.1 ul||1.0 ul||1.0 ul||2.0 ul||5.9 ul||20.0 ul|| |- |#5-3||10.8 ul||1.0 ul||1.0 ul||2.0 ul||5.2 ul||20.0 ul|| |- |#6-6||5.0 ul||1.0 ul||1.0 ul||2.0 ul||11.0 ul||20.0 ul|| |- |#8-15||16.0 ul||1.0 ul||1.0 ul||2.0 ul||-||20.0 ul|| |}
37˚C 3h
{| border="1" class="wikitable" !Digestion pSB1C3 T7-10 |- |||DNA||BamHI-HF (NEB)||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total|| |- |#4-1||4.8 ul||1.0 ul||1.0 ul||2.0 ul||11.2 ul||20.0 ul|| |}
1ul rSAP / 37˚C 1h
{| border="1" class="wikitable" !Ligation |- |||Vector (pSB1C3)||Insert||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total|| |- |#4||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul|| |- |#5||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul|| |- |#6||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul|| |- |#8||1.0 ul||1.0 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul|| |}
22˚C 2h
-80 ˚C 20’’ Heat Inaktivation
Colony PCR of “pTRE - #13 mLacI-miR373 BS” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||CMV fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |22X||55.0 ul||55.0 ul||22.0 ul||22.0 ul||11.0 ul||4.4 ul||270.6 ul||||440.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Results: negative
Mini Prep of “pSB1C3 #6-25,26” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/20/2015 10:01:33 AM||0.4||ng/ul||0.007||-0.003||-2.12||-1.13||DNA||50.00 |- |2||psb1c3 #6-25||biospec||8/20/2015 10:02:45 AM||41.0||ng/ul||0.820||0.421||1.95||1.93||DNA||50.00 |- |3||psb1c3 #6-26||biospec||8/20/2015 10:03:50 AM||46.0||ng/ul||0.920||0.464||1.98||1.97||DNA||50.00 |}
Cut Check of “pSB1C3 #6-25,26” {| border="1" class="wikitable" !Cut Check |- |||DNA ||EcoRI-HF||PstI-HF||Cut Smart Buffer||ddH₂O||Total |- |#6-25||6.1 ul||0.5 ul||0.5 ul||2.0 ul||10.9 ul||20.0 ul |- |#6-26||5.5 ul||0.5 ul||0.5 ul||2.0 ul||11.5 ul||20.0 ul |}
37˚C 3h
Result: negative
Digestion of “pTRE #12” {| border="1" class="wikitable" !Digestion |- |||pTRE #12 (5000 ng/ul)||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total |- |1x||16.0 ul||0.5 ul||0.5 ul||2.0 ul||1.0 ul||20.0 ul |}
37˚C 3h
Result: negative
Digestion of “Damp-Pex pSB1C3” {| border="1" class="wikitable" !Digestion pSB1C3 T7-10 |- |||DNA (500 ng/ul)||NotI||Cut Smart Buffer||ddH₂O||Total|||| |- |#9-5||3.42 ul||1.0 ul||2.0 ul||13.58 ul||20.0 ul|||| |- |#9-10||3.56 ul||1.0 ul||2.0 ul||13.44 ul||20.0 ul|||| |}
37˚C 3h
Ligation of “Damp-Pex pColA” {| border="1" class="wikitable" !Ligation |- |||pColA (100 ng/ul)||Insert (36 ng/ul)||T4 DNA Ligase Buffer||T4 DNA Ligase||ddH₂O||Total|| |- |1x||2.8 ul||5.41 ul||2.0 ul||1.0 ul||15.0 ul||20.0 ul|| |}
22˚C 1.30h
Transformation with NEB5α.
Colony PCR of “Damp-Pex + pColA” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |26X||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||5.2 ul||319.8 ul||||520.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: #5-4, #5-9 Liquid Culture was made.
Mini Prep of “pTetoff #14-4,5,8” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/22/2015 11:01:09 AM||0.1||ng/ul||0.002||-0.008||-0.23||-0.10||DNA||50.00 |- |2||ptetoff 14-4||biospec||8/22/2015 11:02:05 AM||159.6||ng/ul||3.193||1.678||1.90||2.00||DNA||50.00 |- |3||ptetoff 14-5||biospec||8/22/2015 11:03:21 AM||141.1||ng/ul||2.822||1.466||1.93||1.97||DNA||50.00 |- |4||ptetoff 14-8||biospec||8/22/2015 11:04:14 AM||367.5||ng/ul||7.350||3.969||1.85||2.11||DNA||50.00 |}
{| border="1" class="wikitable" !Cut Check I |- |||DNA (250 ng/ul)||SalI (FD)||HindIII (FD)||Fast Digest Buffer||ddH₂O||Total |- |#14-4||1.6 ul||0.5 ul||0.5 ul||2.0 ul||15.4 ul||20.0 ul |- |#14-5||1.8 ul||0.5 ul||0.5 ul||2.0 ul||15.2 ul||20.0 ul |- |#14-8||0.7 ul||0.5 ul||0.5 ul||2.0 ul||16.3 ul||20.0 ul |}
37˚C 2h
Result: negative
{| border="1" class="wikitable" !Cut Check II |- |||DNA (250 ng/ul)||SalI (FD)||NotI (FD)||Fast Digest Buffer||ddH₂O||Total |- |#14-4||1.6 ul||0.5 ul||0.5 ul||2.0 ul||15.4 ul||20.0 ul |- |#14-5||1.8 ul||0.5 ul||0.5 ul||2.0 ul||15.2 ul||20.0 ul |- |#14-8||0.7 ul||0.5 ul||0.5 ul||2.0 ul||16.3 ul||20.0 ul |}
37˚C 30’’
Result: negative
Colony PCR of “pSB1C3 – T7 #4,5,6,8” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |50x||125.0 ul||125.0 ul||50.0 ul||50.0 ul||25.0 ul||10.0 ul||615.0 ul||||1000.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Digestion of “pTRE
#12-2” {| border="1" class="wikitable" !Digestion
|- |||pTRE #12-2 (2000
ng/ul)||EcoRI-HF||XhoI-NEB||Cut Smart
Buffer||ddH₂O||Total|| |- |1x||6.4 ul||0.5
ul||0.5 ul||2.0 ul||10.6 ul||20.0 ul|| |}
37˚C 2h
Result: Bands were at the expected section. Gel Extraction was made.
Mini Prep {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/24/2015 1:28:49 AM||0.0||ng/ul||0.001||-0.013||-0.07||-0.07||DNA||50.00 |- |2||ptre gel||biospec||8/24/2015 1:30:29 AM||15.3||ng/ul||0.306||0.164||1.87||0.65||DNA||50.00 |}
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/24/2015 1:26:27 AM||0.7||ng/ul||0.015||0.001||18.47||0.53||DNA||50.00 |- |2||ptre||biospec||8/24/2015 1:29:38 AM||13.7||ng/ul||0.275||0.145||1.90||0.10||DNA||50.00 |}
Digestion of “TEV-Protease pET45/pSB1C3 T7/pColA {| border="1" class="wikitable" !Digestion |- |||DNA (500 ng/ul)||BamHI-HF||XhoI-HF||Cut Smart Buffer||ddH₂O||Total|| |- |1x||5.86 ul||0.5 ul||0.5 ul||2.0 ul||11.14 ul||20.0 ul|| |}
37˚C 3h
Ligation of “pCAGop with pTRE #12-2” {| border="1" class="wikitable" !Ligation |- |||pCAG (18.38 ng/ul)||pTRE #12-2 (100 ng/ul)||T4 DNA Ligase||T4 DNA Ligase Buffer||Cut Smart Buffer||ddH₂O||Total |- |1x||0.6 ul||6.6 ul||1.0 ul||2.0 ul||2.0 ul||9.8 ul||20.0 ul |}
Transformation was made.
Cut Check “pSB1C3-T7 #4,5,6,8” {| border="1" class="wikitable" !Cut Check!!!!!!!!!!!! |- |||DNA (300 ng/ul)||EcoRI (FD)||PstI (FD)||Fast Digest Buffer||ddH₂O||Total |- |1x||3.5 ul||1.0 ul||1.0 ul||2.0 ul||12.5 ul||20.0 ul |}
30’ digestion
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/25/2015 1:26:12 PM||0.8||ng/ul||0.016||-0.003||-5.85||2.25||DNA||50.00 |- |2||#9-5-4||biospec||8/25/2015 1:27:29 PM||59.1||ng/ul||1.182||0.603||1.96||2.25||DNA||50.00 |- |3||#9-5-9||biospec||8/25/2015 1:28:30 PM||37.9||ng/ul||0.758||0.375||2.02||2.19||DNA||50.00 |}
Colony PCR of “pTRE #13” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||CMV fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |7x||17.5 ul||17.5 ul||7.0 ul||7.0 ul||3.5 ul||1.4 ul||86.1 ul||||140.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Colony PCR of “pCAG-DsRed” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |14x||35.0 ul||35.0 ul||14.0 ul||14.0 ul||7.0 ul||2.8 ul||172.2 ul||||280.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Bands weren’t at the expected section.
Cut Check of „pSB1C3 – TlpB“ {| border="1" class="wikitable" !Cut Check |- |||DNA (250 ng/ul)||EcoRI (FD)||Fast Digest Buffer||ddH₂O||Total|| |- |#8-1||3.6 ul||0.5 ul||2.0 ul||13.9 ul||20.0 ul|| |- |#8-3||3.1 ul||0.5 ul||2.0 ul||14.4 ul||20.0 ul|| |}
7˚C 35’
The colonies will be made glycerol stock.
Ligation of “Damp-Pex” {| border="1" class="wikitable" !Ligation |- |||DNA (500 ng/ul)||NotI||Cut Smart Buffer||ddH₂O||Total|||| |- |#9-5||3.78 ul||1.0 ul||2.0 ul||13.22 ul||20.0 ul|||| |- |#9-10||3.87 ul||1.0 ul||2.0 ul||13.13 ul||20.0 ul|||| |}
37˚C 3h
{| border="1" class="wikitable" !Ligation |- |||ColA (100 ng/ul)||Insert (36 ng/ul)||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total|| |- |#9-5||10.0 ul||14.4 ul||1.0 ul||3.0 ul||1.6 ul||30.0 ul|| |}
22˚C 1.5h
Transformation was made.Colony PCR of “pCAGop-DsRed” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |10x||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||200.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: 2 colonie will be made liquid culture and streak plate. Ligation and transformation will be repeated.
Mini Prep of “pTRE #13-3,5” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/26/2015 4:55:28 PM||0.1||ng/ul||0.003||-0.020||-0.13||-0.13||DNA||50.00 |- |2||ptre 13-3||biospec||8/26/2015 4:56:47 PM||77.9||ng/ul||1.558||0.772||2.02||1.67||DNA||50.00 |- |3||ptre 13-5 ||biospec||8/26/2015 4:58:06 PM||76.0||ng/ul||1.520||0.776||1.96||1.48||DNA||50.00 |}
Colony PCR of “pColA – Damp-Pex, pSB1C3 – TEV {| border="1" class="wikitable" !Colony PCR “pColA – Damp-Pex” |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |10x||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||200.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Colony PCR “pSB1C3 – TEV Protease” {| border="1" class="wikitable" !Colony PCR “pSB1C3 – TEV Protease” |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |10x||25.0 ul||25.0 ul||10.0 ul||10.0 ul||5.0 ul||2.0 ul||123.0 ul||||200.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Colony PCR “pSB1C3 – TEV Protease” – Repeat {| border="1" class="wikitable" !Colony PCR “pSB1C3 – TEV Protease” |- |||MgCl₂||(NH₄)2SO₄||VR||VF||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |15x||37.5 ul||37.5 ul||15.0 ul||15.0 ul||7.5 ul||3.0 ul||184.5 ul||||300.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Colony PCR of “pTRE – Luc #14” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||pTRE Luc rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |14x||35.0 ul||35.0 ul||14.0 ul||14.0 ul||7.0 ul||2.8 ul||172.2 ul||||280.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Bands weren’t at the expected section.
Gel Extraction of “Damp-Pex” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/27/2015 9:39:17 PM||1.1||ng/ul||0.022||0.009||2.36||1.00||DNA||50.00 |- |2||damp-pex||biospec||8/27/2015 9:40:04 PM||8.2||ng/ul||0.164||0.090||1.82||0.05||DNA||50.00 |}
Colony PCR of “pCAGop-DsRed #12” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |14x||35.0 ul||35.0 ul||14.0 ul||14.0 ul||7.0 ul||2.8 ul||172.2 ul||||280.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Negative.
Ligation of “pCAGop-DsRed” {| border="1" class="wikitable" !Ligation |- |||pCAG (18.38 ng/ul)||pTRE #12 (100 ng/ul)||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total |- |1x||0.3 ul||6.6 ul||1.0 ul||2.0 ul||10.1 ul||20.0 ul |}
22˚C 1.30h
Transformation was made.
Colony PCR of „pSB1C3-T7 #5“ {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |8x||20.0 ul||20.0 ul||8.0 ul||8.0 ul||4.0 ul||1.6 ul||98.4 ul||||160.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: #1-1 Liquid Culture was made.
Digestion of “pSB1C3-T7 TEV Protease (17. and 18. Colony)” {| border="1" class="sortable" !Digestion |- |||DNA (500 ng/ul)||NotI-HF||Cut Smart Buffer||ddH₂O||Total |- |17||6.0 ul||0.5 ul||2.0 ul||11.5 ul||20.0 ul |- |18||13.0 ul||0.5 ul||2.0 ul||4.5 ul||20.0 ul |}
37˚C 3h
65˚C 20’’ Heat Inactivation
Result: negative
Digestion of “pColA” {| border="1" class="wikitable" !Digestion |- |||pColA (300 ng/ul)||NotI||Buffer||ddH₂O||Total |- |1x||11.76 ul||0.5 ul||2.0 ul||5.74 ul||20.0 ul |}
37˚C 3h
1.2 ul rSAP
37˚C 1h
20’’ Heat Inactivation
Colony PCR of “pCAGop-DsRed #12-2. Colony streak” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |12x||30.0 ul||30.0 ul||12.0 ul||12.0 ul||6.0 ul||2.4 ul||147.6 ul||||240.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Negative
Cut Check of „pSB1C3-T7 #5-31“ {| border="1" class="wikitable" !Cut Check |- |||DNA||EcoRI (FD)||PstI (FD)||Fast Digest Buffer||ddH₂O||Total |- |1x||3.0 ul||1.0 ul||1.0 ul||2.0 ul||13.0 ul||20.0 ul |} Digestion of “pSB1C3 #9” {| border="1" class="wikitable" !Digestion |- |||DNA (500 ng/ul)||NotI||Cut Smart Buffer||ddH₂O||Total|| |- |1x||3.0 ul||1.0 ul||2.0 ul||14.0 ul||20.0 ul|| |}
3h was digested
Result: #9-5 Bands were at the expected section
Ligation of “pColA #9” {| border="1" class="wikitable" !Ligation |- |||pColA||#9||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total |- |1x||7.5 ul||9.2 ul||1.0 ul||2.0 ul||0.3 ul||20.0 ul |}
2h was ligasted
Mini Prep of “pCAG-DsRed #12” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/28/2015 7:23:31 PM||0.5||ng/ul||0.010||-0.006||-1.58||-1.46||DNA||50.00 |- |2||pcag op-dsred||biospec||8/28/2015 7:24:38 PM||92.3||ng/ul||1.846||1.105||1.67||0.79||DNA||50.00 |- |3||pcag op-dsred||biospec||8/28/2015 7:26:20 PM||67.4||ng/ul||1.348||0.702||1.92||1.35||DNA||50.00 |}
Cut Check “pCAGop-DsRed #12” {| border="1" class="wikitable" !Cut Check |- |||DNA||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total |- |1x||3.7 ul||0.5 ul||0.5 ul||2.0 ul||13.3 ul||20.0 ul |}
Result: negative
Cut Check “pCAGop-DsRed #12” - Repeat {| border="1" class="wikitable" !Cut Check |- |||DNA||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total |- |1x||3.7 ul||0.5 ul||0.5 ul||2.0 ul||13.3 ul||20.0 ul |}
Result: negative
Colony PCR of „pTEToff and pTEToff N term“ (28.08.2015) {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||CMV rv||pTRE Luc fw||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |26x||65.0 ul||65.0 ul||26.0 ul||26.0 ul||13.0 ul||5.2 ul||319.8 ul||||520.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: positive
Mini Prep of “pSB1C3-T7 TEV Protease” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/28/2015 5:55:09 PM||0.0||ng/ul||-0.001||-0.010||0.10||-1.29||DNA||50.00 |- |2||tev psb1c3-t7/17||biospec||8/28/2015 5:56:45 PM||81.8||ng/ul||1.637||0.848||1.93||2.08||DNA||50.00 |- |3||tev psb1c3-t7/18||biospec||8/28/2015 5:59:01 PM||38.5||ng/ul||0.771||0.380||2.03||2.03||DNA||50.00 |}
Mini Prep of “pSB1C3-T7 TEV Protease” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/29/2015 8:08:11 PM||-0.8||ng/ul||-0.016||-0.019||0.83||0.68||DNA||50.00 |- |2||psb1c3-t7/tev17||biospec||8/29/2015 8:09:26 PM||135.4||ng/ul||2.709||1.355||2.00||1.68||DNA||50.00 |- |3||psb1c3-t7/tev18||biospec||8/29/2015 8:11:06 PM||229.1||ng/ul||4.581||2.193||2.09||2.40||DNA||50.00 |}
Mini Prep of “pCAGop-DsRed #1-1” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/30/2015 11:05:33 AM||0.2||ng/ul||0.003||-0.012||-0.25||-0.27||DNA||50.00 |- |2||pcag op-dsred||biospec||8/30/2015 11:07:31 AM||79.8||ng/ul||1.595||0.823||1.94||1.72||DNA||50.00 |}
Cut Check of “pCAGop-DsRed #1-1” {| border="1" class="wikitable" !Cut Check |- |||pCAGop-DsRed (250 ng/ul)||EcoRI-HF||XhoI (NEB)||Cut Smart Buffer||ddH₂O||Total |- |1x||3.2 ul||0.5 ul||0.5 ul||2.0 ul||13.8 ul||20.0 ul |}
37˚C 3h
Result: negative
Digestion of “pSB1C3-T7 TEV Protease” {| border="1" class="wikitable" !Digestion |- |||Insert||NotI||Cut Smart Buffer||ddH₂O||Total |- |17||3.7 ul||0.25 ul||2.0 ul||14.05 ul||20.0 ul |- |18||2.2 ul||0.25 ul||2.0 ul||15.55 ul||20.0 ul |}
37˚C 3h
Result: Bands were at the expected section. Gel extraction was made.
Ligation of “pSB1C3-T7 TEV Protease” {| border="1" class="wikitable" !Ligation |- |||Vector (60 ng/ul)||Insert (29.11 ng/ul)||T4 DNA Buffer||T4 DNA Buffer Ligase||ddH₂O||Total |- |1x||7.0 ul||8.7 ul||2.0 ul||1.0 ul||1.3 ul||20.0 ul |}
22˚C 1.5h
Transformation was made.
Gel Extraction {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||8/30/2015 5:28:51 PM||0.0||ng/ul||-0.001||-0.005||0.15||0.03||DNA||50.00 |- |2||psb1c3-t7||biospec||8/30/2015 5:28:05 PM||5.6||ng/ul||0.113||0.061||1.85||0.17||DNA||50.00 |}
Colony PCR of “pColA – TEV” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |16x||40.0 ul||40.0 ul||16.0 ul||16.0 ul||8.0 ul||3.2 ul||196.8 ul||||320.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Colony PCR of “pColA – TEV” – Repeat {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pColA fw||pColA rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |22x||55.0 ul||55.0 ul||22.0 ul||22.0 ul||11.0 ul||3.2 ul||270.6 ul||||440.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Digestion of “PsicA for pSB1C3 and invF-sicA for ColA” (31.08.2015) {| border="1" class=wikitable" !Digestion of “PsicA and invF” |- |||DNA||EcoRI-HF||PstI||XbaI||SpeI||2.1 Buffer||Cut Smart Buffer||ddH₂O||Total |- |invF-sicA||10.0 ul||-||-||1.0 ul||1.0 ul||-||2.0 ul||6.0 ul||20.0 ul |- |PsicA||10.0 ul||1.0 ul||1.0 ul||-||-||2.0 ul||-||6.0 ul||20.0 ul |}
37˚C 10h
82˚C 20’ Heat Inactivation
{| border="1" class="wikitable" !Digestion of “pSB1C3-RFP and pColA-NotI” |- |||DNA||EcoRI (FD)||PstI (FD)||XbaI (FD)||SpeI (FD)||Fast Digest Buffer||ddH₂O||Total|| |- |pSB1C3-RFP (1000 ng/ul)||2.5 ul||1.0 ul||1.0 ul||-||-||2.0 ul||13.5 ul||20.0 ul|| |- |pColA-NotI (500 ng/ul)||5.0 ul||-||-||1.0 ul||1.0 ul||2.0 ul||11.0 ul||20.0 ul|| |}
September
22˚C 2h
{| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/1/2015 4:10:17 AM||0.3||ng/ul||0.006||-0.007||-0.88||-0.78||DNA||50.00 |- |2||colA ext||biospec||9/1/2015 4:11:28 AM||11.7||ng/ul||0.235||0.132||1.78||0.08||DNA||50.00 |- |3||cola clean-up||biospec||9/1/2015 4:12:56 AM||7.2||ng/ul||0.143||0.078||1.84||0.06||DNA||50.00 |- |4||psb1c3 ext||biospec||9/1/2015 4:14:40 AM||10.6||ng/ul||0.213||0.113||1.88||0.12||DNA||50.00 |}
Transformation will be made.
Ligation of “pCAGop-DsRed” {| border="1" class=wikitable" !Ligation |- |||pCAG||pTRE #12||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total|| |- |1x||0.3 ul||4.0 ul||1.0 ul||1.0 ul||13.7 ul||20.0 ul|| |}
Transformation with BL21.
Digestion of “pSB1C3-T7 TEV and pColA {| border="1" class="wikitable" !Digestion |- |||DNA||XhoI-HF||SpeI-HF||Cut Smart Buffer||RNAse||ddH₂O||Total |- |17 (Hand Made Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||0.2 ul||7.3 ul||20.0 ul |- |18 (Hand Made Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||0.2 ul||7.3 ul||20.0 ul |- |17 (Midi Prep Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||-||7.5 ul||20.0 ul |- |18 (Midi Prep Solution)||10.0 ul||0.25 ul||0.25 ul||2.0 ul||-||7.5 ul||20.0 ul |- |pColA (1000 ng/ul)||9.0 ul||0.5 ul||0.5 ul||2.0 ul||-||8.0 ul|| |}
37˚C 3h
80˚C 20’ Heat Inactivation
Cut Check was made.
Result: pColA Bands were at the expected section. Midi Prep Solution is better.
Colony PCR of “pCAGop-DsRed and pCMV-lacO” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |16x||40.0 ul||40.0 ul||16.0 ul||16.0 ul||8.0 ul||3.2 ul||196.8 ul||||320.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative. It will be repeated.
Colony PCR of “invF-sicA and PsicA” (02.09.2015)
Result: PsicA: 1, 2, 6, 8, 9, 10, 11 Liquid Culture will be made.
Digestion of “invF-sicA for pSB1C3 and ColA”
DNA 10.0 ul + rSAP 1.0 ul / 37˚C 1h / 80˚C 20’
{| border="1" class="wikitable" !Digestion |- |||DNA||EcoRI-HF||PstI||XbaI||SpeI||2.1 Buffer||Cut Smart Buffer||ddH₂O||Total |- |pSB1C3||10.0 ul||0.4 ul||0.4 ul||-||-||2.0 ul||-||7.2 ul||20.0 ul |- |ColA||10.0 ul||-||-||0.4 ul||0.4 ul||-||2.0 ul||7.2 ul||20.0 ul |}37˚C overnight
Mini Prep of “PsicA – pSB1C3” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/3/2015 8:34:29 PM||0.8||ng/ul||0.016||0.000||116.17||0.85||DNA||50.00 |- |2||psb1c3-psica 1||biospec||9/3/2015 8:35:32 PM||130.5||ng/ul||2.610||1.370||1.91||2.04||DNA||50.00 |- |3||psb1c3-psica 2||biospec||9/3/2015 8:36:34 PM||140.9||ng/ul||2.818||1.457||1.93||2.16||DNA||50.00 |- |4||psb1c3-psica 6||biospec||9/3/2015 8:37:28 PM||178.5||ng/ul||3.569||1.864||1.92||2.20||DNA||50.00 |- |5||psb1c3-psica 8||biospec||9/3/2015 8:38:24 PM||98.7||ng/ul||1.974||1.021||1.93||2.13||DNA||50.00 |- |6||psb1c3-psica 9||biospec||9/3/2015 8:39:21 PM||237.0||ng/ul||4.741||2.476||1.91||2.13||DNA||50.00 |- |7||psb1c3-psica 10||biospec||9/3/2015 8:40:35 PM||109.0||ng/ul||2.181||1.127||1.94||2.05||DNA||50.00 |- |8||||biospec||9/3/2015 8:41:51 PM||109.5||ng/ul||2.189||1.134||1.93||1.97||DNA||50.00 |- |9||blank||biospec||9/3/2015 8:43:10 PM||1.3||ng/ul||0.025||0.003||9.26||0.73||DNA||50.00 |- |10||psb1c3-psica 11||biospec||9/3/2015 8:44:14 PM||50.4||ng/ul||1.008||0.518||1.95||1.97||DNA||50.00 |}
Cut Check of “PsicA – pSB1C3” {| border="1" class="wikitable" !Cut Check |- |||DNA||EcoRI (FD)||PstI (FD)||Fast Digest Buffer||ddH₂O||Total |- |1||1.9 ul||0.35 ul||0.35 ul||2.0 ul||15.4 ul||20.0 ul |- |2||1.8 ul||0.35 ul||0.35 ul||2.0 ul||15.5 ul||20.0 ul |- |6||1.4 ul||0.35 ul||0.35 ul||2.0 ul||15.9 ul||20.0 ul |- |8||2.5 ul||0.35 ul||0.35 ul||2.0 ul||14.8 ul||20.0 ul |- |9||1.1 ul||0.35 ul||0.35 ul||2.0 ul||16.2 ul||20.0 ul |- |10||2.3 ul||0.35 ul||0.35 ul||2.0 ul||15.0 ul||20.0 ul |- |11||5.0 ul||0.35 ul||0.35 ul||2.0 ul||12.3 ul||20.0 ul |}
37˚C 35’
Ligation of “invF and pSB1C3/pColA” {| border="1" class="wikitable" !Ligation!!!!!!!!!!!!!! |- |||Vector (100 ng/ul)||invF/sicA||invF/sicA||T4 DNA Ligase||T4 DNA Ligase Buffer||ddH₂O||Total |- |ColA (-)||11.6 ul||-||-||1.0 ul||3.2 ul||16.6 ul||32.2 ul |- |ColA (+)||11.6 ul||14.40 ul||-||3.2 ul||3.4 ul||2.2 ul||34.8 ul |- |pSB1C3||9.4 ul||-||14.40 ul||1.0 ul||3.0 ul||2.2 ul||30.0 ul |}
RT 1h
Transformation will be made.
Colony PCR of “pCMV-Lacop-DsRed and pCAGop-DsRed” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |15x||37.5 ul||37.5 ul||15.0 ul||15.0 ul||7.5 ul||3.0 ul||184.5 ul||||300.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: Bands were at the expected section. Liquid Culture was made.
Mini Prep of „pCMV-Lacop-DsRed and pCAGop-DsRed“ {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/5/2015 10:42:13 AM||-0.1||ng/ul||-0.003||-0.015||0.18||0.15||DNA||50.00 |- |2||pcmv-lacop-dsred 1||biospec||9/5/2015 10:44:11 AM||68.4||ng/ul||1.369||0.690||1.98||1.76||DNA||50.00 |- |3||pcmv-lacop-dsred 2||biospec||9/5/2015 10:45:12 AM||51.2||ng/ul||1.024||0.499||2.05||1.77||DNA||50.00 |- |4||pcmv-lacop-dsred 3||biospec||9/5/2015 10:46:09 AM||78.5||ng/ul||1.570||0.793||1.98||1.98||DNA||50.00 |- |5||pcmv-lacop-dsred 4||biospec||9/5/2015 10:47:04 AM||47.7||ng/ul||0.954||0.460||2.07||1.95||DNA||50.00 |- |6||Pcagop-dsred 1||biospec||9/5/2015 10:48:08 AM||499.6||ng/ul||9.991||5.290||1.89||2.20||DNA||50.00 |}
Cut Check of „pCMV-Lacop-DsRed and pCAGop-DsRed“ {| border="1" class="wikitable" !Cut Check |- |||DNA||BamHI||XhoI||EcoRI||Cut Smart Buffer||ddH₂O||Total |- |1||4.4 ul||0.5 ul||0.5 ul||-||2.0 ul||12.6 ul||20.0 ul |- |2||5.9 ul||0.5 ul||0.5 ul||-||2.0 ul||11.1 ul||20.0 ul |- |3||3.9 ul||0.5 ul||0.5 ul||-||2.0 ul||13.1 ul||20.0 ul |- |4||6.3 ul||0.5 ul||0.5 ul||-||2.0 ul||10.7 ul||20.0 ul |- |pCAGop-DsRed||1.0 ul||0.5 ul||-||0.5 ul||2.0 ul |- |||16.0 ul||20.0 ul |}
37˚C 3h
Result: 1-2-3-4 is positive.
Cut Check of “invF
and sicA” {| border="1" class="wikitable" !Cut
Check |- |||DNA (250 ng/ul)||EcoRI (FD)||PstI
(FD)||Fast Digest Buffer||ddH₂O||Total|| |-
|1x||5.0 ul||0.5 ul||0.5 ul||2.0 ul||12.0
ul||20.0 ul|| |}
37˚C 35’
Mini Prep {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/7/2015 6:34:56 PM||-0.1||ng/ul||-0.003||-0.005||0.60||0.11||DNA||50.00 |- |2||#18-11||biospec||9/7/2015 6:36:20 PM||79.8||ng/ul||1.597||0.843||1.89||1.93||DNA||50.00 |- |3||#18-2||biospec||9/7/2015 6:37:48 PM||74.9||ng/ul||1.497||0.787||1.90||2.01||DNA||50.00 |- |4||elution blank ||biospec||9/7/2015 6:39:44 PM||0.3||ng/ul||0.005||-0.004||-1.51||0.68||DNA||50.00 |- |5||#18-5 elution||biospec||9/7/2015 6:40:49 PM||57.7||ng/ul||1.154||0.604||1.91||1.91||DNA||50.00 |}
Digestion of “#18-2,5” {| border="1" class="wikitable" !Digestion |- |||DNA (500 ng/ul)||XbaI||SpeI-HF||Cut Smart Buffer||ddH₂O||Total |- |#18-2||6.66 ul||0.4 ul||0.4 ul||2.0 ul||8.54 ul||20.0 ul |- |#18-5||8.66 ul||0.4 ul||0.4 ul||2.0 ul||10.54 ul||20.0 u |}
37˚C 40’
Ligation {| border="1" class="wikitable" !Ligation |- |||pSB1C3-invF (175 ng/ul)||ColA (100 ng/ul)||T4 DNA Ligase ||T4 DNA Ligase Buffer||ddH₂O||Total |- |1||7.0 ul||-||1.0 ul||2.0 ul||9.17 ul||20.0 ul |- |2||-||7.0 ul||1.0 ul||2.0 ul||5.0 ul||20.0 ul |}
Transformation will be made.
Mini Prep of “pSB1C3 – TEV” {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/8/2015 10:39:08 AM||-0.1||ng/ul||-0.003||-0.009||0.30||0.13||DNA||50.00 |- |2||psb1c3-tev 17||biospec||9/8/2015 10:40:15 AM||125.5||ng/ul||2.510||1.331||1.89||2.16||DNA||50.00 |- |3||psb1c3-tev 18||biospec||9/8/2015 10:42:45 AM||111.9||ng/ul||2.238||1.195||1.87||2.14||DNA||50.00 |}
Digestion of “pSB1C3 – TEV 17/18 and pColA-Toehold” {| border="1" class="wikitable" !Digestion of “pSB1C3 – TEV 17/18” |- |||DNA (1000 ng/ul)||XbaI (FD)||SpeI (FD)||Fast Digest Buffer||ddH₂O||Total |- |17||8.0 ul||1.0 ul||1.0 ul||2.0 ul||8.0 ul||20.0 ul |- |18||9.0 ul||1.0 ul||1.0 ul||2.0 ul||7.0 ul||20.0 ul |}
37˚C 1h
{| border="1" class="wikitable" !Digestion of “pColA-Toehold” |- |||DNA (1000 ng/ul)||XbeI (FD)||SpeI (FD)||Fast Digest Buffer||ddH₂O||Total |- |1x||16.0 ul||1.0 ul||1.0 ul||2.0 ul||-||20.0 ul |}
37˚C 1h
Colony PCR of “ pSB1C3-TEV 17/18” {| border="1" class="wikitable" !Colony PCR |- |||MgCl₂||(NH₄)2SO₄||pTRE Luc fw||SV40 rv||dNTP||Tag||ddH₂O||DNA||Total |- |1x||2.5 ul||2.5 ul||1.0 ul||1.0 ul||0.5 ul||0.2 ul||12.3 ul||5.0 ul||25.0 ul |- |25x||61.5 ul||61.5 ul||25.0 ul||25.0 ul||12.5 ul||5.0 ul||307.5 ul||||498.0 ul |} {| border="1" class="wikitable" !Cycling |- |||95˚C||95˚C||56˚C||72˚C||72˚C||Cycle |- |Time||5’||30’’||30’’||1.5’||5’||35x |}
Result: negative
Digestion of “pColA / pSB1C3 #9 / pColA-Toehold #2-8 / pSB1C3 T7 #10-17 / #18” {| border="1" class="wikitable" !Digestion of |- |||DNA (1000 ng/ul)||XbaI (FD)||SpeI (FD)||Cut Smart Buffer||ddH₂O||Total |- |pColA||4.5 ul||0.5 ul||0.5 ul||2.0 ul||12.5 ul||20.0 ul |- |pSB1C3 #9||1.7 ul||0.5 ul||0.5 ul||2.0 ul||15.3 ul||20.0 ul |- |pColA-Toehold #2-8||16.0 ul||0.5 ul||0.5 ul||2.0 ul||1.0 ul||20.0 ul |- |pSB1C3 T7 #10-17||3.3 ul||0.5 ul||0.5 ul||2.0 ul||13.7 ul||20.0 ul |- |#18||3.0 ul||0.5 ul||0.5 ul||2.0 ul||14.0 ul||20.0 ul |}
37˚C 3h<7p>
pColA-Toehold #2-8: 1ul rSAP / 37˚C 1h / 80˚C 20’
Result: pColA and #10 gel extraction will be made.
Gel extraction {| border="1" class=wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/10/2015 7:29:27 PM||-0.2||ng/ul||-0.003||-0.009||0.37||0.28||DNA||50.00 |- |2||psb1c3-#9||biospec||9/10/2015 7:31:01 PM||308.0||ng/ul||6.160||3.182||1.94||2.10||DNA||50.00 |} {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/10/2015 8:13:13 PM||0.3||ng/ul||0.005||-0.003||-1.70||-2.25||DNA||50.00 |- |2||psb1c3 t7-10 (17)||biospec||9/10/2015 8:14:54 PM||152.2||ng/ul||3.045||1.612||1.89||2.25||DNA||50.00 |- |3||psb1c3 t7-10 (18)||biospec||9/10/2015 8:16:02 PM||164.6||ng/ul||3.291||1.737||1.90||2.23||DNA||50.00 |} {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/11/2015 10:05:05 PM||0.1||ng/ul||0.002||-0.017||-0.13||-0.11||DNA||50.00 |- |2||pcola (notI)||biospec||9/11/2015 10:07:08 PM||40.4||ng/ul||0.807||0.399||2.03||1.75||DNA||50.00 |- |3||psb1c3 #9||biospec||9/11/2015 10:08:33 PM||76.0||ng/ul||1.520||0.789||1.93||2.17||DNA||50.00 |- |4||psb1c3-t7 #10||biospec||9/11/2015 10:09:55 PM||71.5||ng/ul||1.431||0.721||1.99||2.15||DNA||50.00 |- |5||blank ||biospec||9/11/2015 10:14:04 PM||-0.4||ng/ul||-0.007||-0.020||0.35||0.32||DNA||50.00 |- |6||psb1c3-t7 #10||biospec||9/11/2015 10:15:01 PM||70.4||ng/ul||1.408||0.727||1.94||2.16||DNA||50.00 |} {| border="1" class="wikitable" !#!!Sample ID!!User name!!Date and Time!!Nucleic Acid Conc.!!Unit!!A260!!A280!!260/280!!260/230!!Sample Type!!Factor |- |1||blank||biospec||9/11/2015 2:24:30 AM||0.6||ng/ul||0.011||0.002||4.95||1.18||DNA||50.00 |- |2||Pcola-2||biospec||9/11/2015 2:26:25 AM||145.0||ng/ul||2.901||1.398||2.08||2.04||DNA||50.00 |}