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 1Gradient 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 |
57-64˚C
Results weren’t matching with expected results, experiment will be repeated.
(30.06.2015)
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.
Gradient PCR from pCAGGS (CAG FWD – CAG 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.
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)
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
98˚C | 98˚C | 56˚C | 72˚C | 72˚C | Cycle | |
Time | 2’ | 10’’ | 30’’ | 1’ | 5’ | 35x |
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
98˚C | 98˚C | 56˚C | 72˚C | 72˚C | Cycle | |
Time | 2’ | 10’’ | 30’’ | 1’ | 5’ | 35x |
Defterde jel görüntüsü yok.
Gradient PCR from pCAGGS | ||||||||
---|---|---|---|---|---|---|---|---|
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 |
62-64˚C
Result: Gel extraction was performed. PCR (+): 680 bp
# | 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”
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.
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”
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.
# | 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
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.
# | 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
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.
Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers” | |||||||||
---|---|---|---|---|---|---|---|---|---|
MgCl₂ | (NH₄)2SO₄ | pTRE Luc fwd | 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 |
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
95˚C | 95˚C | 55˚C | 72˚C | 72˚C | Cycle | |
Time | 5’ | 30’’ | 30’’ | 1.5’ | 5’ | 35x |
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
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.
Colony PCR from “pCAG” with “pTRE Luc fwd/SV40 polyA re. primers” | |||||||||
---|---|---|---|---|---|---|---|---|---|
MgCl₂ | (NH₄)2SO₄ | pTRE Luc fwd | 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 |
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
95˚C | 95˚C | 55˚C | 72˚C | 72˚C | Cycle | |
Time | 5’ | 30’’ | 30’’ | 1.5’ | 5’ | 35x |
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
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
98˚C | 98˚C | 64/68˚C | 72˚C | 72˚C | Cycle | |
Time | 2’ | 10’’ | 30’’ | 1’ | 5’ | 35x |
Gel Extraction will made.
# | 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.
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 fmol
PCR of “G-Blocks from IDT” (24.07.2015)
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 |
Cycling for 3x | ||||||
---|---|---|---|---|---|---|
95˚C | 95˚C | 57˚C | 72˚C | 72˚C | Cycle | |
Time | 5’ | 30’’ | 30’’ | 1.5’ | 5’ | 35x |
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
# | 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“
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.)
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”
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# | 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”
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 |
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)
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 |
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 |
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 |
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 |
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
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
95˚C | 95˚C | 60˚C | 72˚C | 72˚C | Cycle | |
Time | 5’ | 30’’ | 2’ | 1.5’ | 5’ | 35x |
Result: negative
# | 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”
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 |
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”
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.
# | 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)
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)“
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
95˚C | 95˚C | 55˚C | 72˚C | 72˚C | Cycle | |
Time | 5’ | 30’’ | 2’ | 1.5’ | 5’ | 35x |
</html>
Result: negative
GEL GÖRÜNTÜSÜ
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”
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 |
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.
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)”
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
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 |
</html> Room Temperature 2h
Creating of “pTRE – mLacI miRNA-BS” (27.07.2015) </html>
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.
GEL GÖRÜNTÜSÜ# | 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)
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)
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 |
# | 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)
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”
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”
# | 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
# | 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
# | 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“
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 |
Cycling | ||||||
---|---|---|---|---|---|---|
95˚C | 95˚C | 56˚C | 72˚C | 72˚C | Cycle | |
Time | 5’ | 30’’ | 30’’ | 1.5’ | 5’ | 35x |
Result: negative
GEL GÖRÜNTÜLERIAugust
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!
# | Sample ID | User name | Date and Time | Nucleic Acid Conc. | Unit | A260 | A280 | 260/280 | 260/230 | Sample Type | Factor |
---|---|---|---|---|---|---|---|---|---|---|---|
1 | blank | biospec | 8/1/2015 6:03:37 PM | -0.1 | ng/ul | -0.003 | -0.009 | 0.30 | 0.12 | DNA | 50.00 |
2 | psb1c3-A | biospec | 8/1/2015 6:06:00 PM | 207.0 | ng/ul | 4.140 | 2.184 | 1.90 | 2.11 | DNA | 50.00 |
3 | psb1c3-B | biospec | 8/1/2015 6:07:07 PM | 209.0 | ng/ul | 4.179 | 2.210 | 1.89 | 2.19 | DNA | 50.00 |
4 | psb1c3-C | biospec | 8/1/2015 6:08:05 PM | 157.3 | ng/ul | 3.147 | 1.664 | 1.89 | 2.09 | DNA | 50.00 |
5 | psb1c3-D | biospec | 8/1/2015 6:08:58 PM | 242.5 | ng/ul | 4.851 | 2.548 | 1.90 | 2.11 | DNA | 50.00 |
6 | psb1c3-E | biospec | 8/1/2015 6:09:42 PM | 116.4 | ng/ul | 2.329 | 1.215 | 1.92 | 2.17 | DNA | 50.00 |
7 | psb1c3-F | biospec | 8/1/2015 6:10:28 PM | 236.4 | ng/ul | 4.729 | 2.503 | 1.89 | 2.17 | DNA | 50.00 |
8 | psb1c3-71 | biospec | 8/1/2015 6:11:33 PM | 111.7 | ng/ul | 2.233 | 1.175 | 1.90 | 2.10 | DNA | 50.00 |
9 | psb1c3-72 | biospec | 8/1/2015 6:12:14 PM | 106.4 | ng/ul | 2.127 | 1.100 | 1.93 | 2.23 | DNA | 50.00 |
10 | psb1c3-8 | biospec | 8/1/2015 6:12:51 PM | 90.7 | ng/ul | 1.814 | 0.959 | 1.89 | 1.98 | DNA | 50.00 |
Ligation of „pSB1C3 – Gblocks“ (02.08.2015)
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 |
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 |
22X | 55.0 ul | 55.0 ul | 22.0 ul | 22.0 ul | 11.0 ul | 4.4 ul | 271.0 ul | 440.4 ul |
Cycling | ||||||
---|---|---|---|---|---|---|
95˚C | 95˚C | 56˚C | 72˚C | 72˚C | Cycle | |
Time | 5’ | 30’’ | 30’’ | 1.5’ | 5’ | 35x |