Team:ATOMS-Turkiye/Extras/Notebook


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
Gradient PCR of „pCAG with CMV-Enhancer FWD/Cβ-Actin REV. Primers” (29.06.2015)

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

57-64˚C

Results weren’t matching with expected results, experiment will be repeated.


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.

Week 2
Gradient PCR of „pCAG with CAG FWD/CAG REV. Primers/pCMV REV.” (01.07.2015)

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.

Week 3
Gradient PCR of „pCAG with CAG-FWD/CAG REV. Primers and Phusion Pol.”

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.

Week 4
Creating “Plasmid pCAG” – Continue

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.

Week 5
Creating “Plasmid pCAG” – Continue

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> <a href="ATOMS-Turkiye_26_07_15_colony_pcr.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_26_07_15_colony_pcr.jpeg" width="390" height="230"></a>

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”

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” </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.


# 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

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!

Week 6
Mini Prep of “pSB1C3-RFP”

# 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“

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


Week 7
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 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

Result: negative


Colony PCR of „pSB1C3 – Gblocks #14“

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
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”

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”

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


# 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)

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


Cycling SV40 rev/CMV fwd
95˚C 95˚C 63˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1’ 2’ 35x


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”

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.


# 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”

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
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”

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”

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”

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
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”

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)

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
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

Result: negative

GEL GÖRÜNTÜSÜ
Colony PCR of “5,6,7,9 – Gblocks”

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
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

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
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”

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.

# 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


Week 8 Ligation of “PET45 – HNS”

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”

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”

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
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”

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
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”

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

22˚C 1h


Mini Prep of “pColA-3,4,5”

# 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)

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

# 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


# 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”

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


# 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”

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
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”

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
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”

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”

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”

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
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”

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


# 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”

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
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 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 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


Week 8 Ligation of “pColA and pSB1C3 #9 (Damp-Pex)” </html>

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”

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
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”

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
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”

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
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”

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
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”

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


# 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”

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”

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”

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
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.


# 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”

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”

# 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)

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


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


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”

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
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”

# 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”

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”

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

<a href="ATOMS-Turkiye_psb1c3_6%2C25%2C26.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_psb1c3_6%2C25%2C26.jpeg" width="410" height="360"></a>

Result: negative


Digestion of “Damp-Pex pSB1C3”

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”

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”

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
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”

# 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


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


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”

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
Cycling
95˚C 95˚C 56˚C 72˚C 72˚C Cycle
Time 5’ 30’’ 30’’ 1.5’ 5’ 35x

</div> 

     <a href="#" class="clickme" id="toggle" onclick="toggle_visibility('8');">Week 8<img              
          src="  LMU14_arrow_down.png" id="8" class="tiger"></a>

Digestion of “pTRE #12-2” </html>

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

# 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


# 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

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”

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

<htm>

Transformation was made.


Cut Check “pSB1C3-T7 #4,5,6,8” </html>

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


# 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”

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
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“

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”

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


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”

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
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”

# 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

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
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”

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
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

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
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”

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
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”

# 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”

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
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”

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“

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
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)”

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”

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”

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
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“

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”

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”

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”

# 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”

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

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)

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
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”

# 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”

# 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”

# 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”

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

</html>

37˚C 3h

<a href="ATOMS-Turkiye_30_08_pcagop_dsred.jpeg" data-lightbox="image-1" class="img="left"><img src="ATOMS-Turkiye_30_08_pcagop_dsred.jpeg" width="410" height="360"></a>

Result: negative


Digestion of “pSB1C3-T7 TEV Protease” </html>

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”

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

# 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”

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
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

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
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)

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


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

02.09.2015 Wiki meetings continue, we are making decisions together. 06.09.2015 What will we be wearing at the Giant Jamboree? Thinking about how to design our T-shirts. 08.09.2015 Team pictures mustn’t be left ends. 11.09.2015 We taught our little friends synthetic biology. Those kids were really funny when they came first, they were covering their faces because they were scared of being contaminated. 13.09.2015 Coloring our drawings is complete. They are better than original now. We still have wiki meetings, there are only 5 days to wiki-freeze!
Ligation of “ColA and pSB1C3”

Ligation
Vector (100 ng/ul) invF-sicA psicA T4 DNA Ligase T4 DNA Ligase Buffer ddH₂O Total
ColA 8.5 ul 17.5 ul - 1.0 ul 2.0 ul - 30.0 ul
pSB1C3 9.44 ul - 13.55 ul 1.0 ul 2.0 ul 3.0 ul 30.0 ul

22˚C 2h


# 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”

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

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”

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
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’

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”

# 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”

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”

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”

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
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“

# 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“

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.

Week 9 Cut Check of “invF and sicA”

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

# 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”

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

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”

# 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”

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


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”

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
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”

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

# 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
# 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
# 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
# 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