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Revision as of 11:53, 17 September 2015


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 Turksih 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.


(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.

Week 2
Gradient PCR of „pCAG with CAG FWD/CAG REV. Primers/pCMV REV.” (01.07.2015) {| border="1" class="wikitable" !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.

GEL GÖRÜNTÜSÜ


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.” (07.07.2015)

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” (09.07.2015)

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” (10.07.2015)

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 (08.07.2015)

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.

GEL GÖRÜNTÜSÜ


# 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 (12.07.2015)

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

Sonuc: 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 (13.07.2015)

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

GEL GÖRÜNTÜSÜ
Creating “Plasmid pCAG” – Repeat (19.07.2015)

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 (20.07.2015)

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

GEL GÖRÜNTÜSÜ
Creating “pCAG (Plasmid) – Repeat (23.07.2015)

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.

GEL GÖRÜNTÜSÜ

# 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” (S. 23/23.7.2015)

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
Digestion of „G-Blocks from IDT and pSB1C3“ (23.07.2015)

Digestion
Insert EcoRI-HF PstI NEB 2.1 Buffer ddH₂O Total
1x 10.0 ul 1.0 ul 1.0 ul 2.0 ul 6.0 ul 20.0 ul
17x 10.0 ul 17.0 ul 17.0 ul 34.0 ul 102.0 ul 20.0 ul
Digestion
Vector EcoRI-HF PstI NEB 2.1 Buffer ddH₂O Total
2x 17.0 ul 1.0 ul 1.0 ul 2.0 ul - 21.0 ul

Result: Gel extraction was made.

GEL GÖRÜNTÜSÜ
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 GÖRÜNTÜSÜ
Gel Extraction (24.07.2015)

# 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“ (24.07.2015)

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” (25.07.2015)

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.

GEL GÖRÜNTÜLERI
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 (25.07.2015)

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

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 7/26/2015 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” (26.07.2015)

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” (26.07.2015)

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.

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 7/26/2015 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” (27.07.2015) {| 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 |}

Sonuc: Only 16-9 is positive.

GEL GÖRÜNTÜLERI
Colony PCR of “pSB1C3 – Gblocks” (28.07.2015)

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

GEL GÖRÜNTÜLERI
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

GEL GÖRÜNTÜSÜ
Colony PCR of „9-10 GBlocks“ (31.07.2015)

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 GÖRÜNTÜLERI

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“ (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


Week 6 Colony PCR of „pSB1C3 – Gblocks“ (03.08.2015)

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