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.
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.
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.
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.
Gradient PCR of „pCAG with CAG-FWD/CAG REV. Primers and Phusion Pol.” (07.07.2015)
62-64˚C Result: Gel extraction was performed. PCR (+): 680 bp Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” (09.07.2015)
pTRE-delta-TRE was made after digestion. Concentration: 99.9 ng/ul The final concentration of pCAG is 6.855 ng/ul. Ligation products were transformed into E.Coli/BL321 strain. Result: No colonies were observed. Result: Bands were at the expected section. Gel extraction was made. GEL GÖRÜNTÜSÜ Creating “Plasmid pCAG” via “pTRE” and “Promoter pCAG” – Repeat (08.07.2015)
Result: Gel extraction was made. GEL GÖRÜNTÜSÜ 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.
Creating “Plasmid pCAG” – Continue (13.07.2015)
Result: All bands were observed around 700 bp, results were negative. Ligation will be repeated. (+) bant: 923 bp (-) bant: 705 bp GEL GÖRÜNTÜSÜ
Vector:Insert 3:1 RT 2h Transformation at BL21. CIP (+): No colonies were absorved. CIP (-): Colony PCR will be made. Creating “Plasmid pCAG” – Continue (20.07.2015)
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)
Gel Extraction will made. GEL GÖRÜNTÜSÜ
Resuspension of “Newly Arrived G-Blocks from IDT” (S. 23/23.7.2015)
100 ul TE for all tubes. Not: 100ng pSB1C3 (2050 bp) ≈ 75 fmol
Digestion of „G-Blocks from IDT and pSB1C3“ (23.07.2015)
Result: Gel extraction was made. GEL GÖRÜNTÜSÜ
PCR of “G-Blocks from IDT” (24.07.2015)
Results: Bands were at the expected section. GEL GÖRÜNTÜSÜ
Gel Extraction (24.07.2015)
Ligation of „G-Blocks from IDT and pSB1C3“ (24.07.2015)
RT 1h Transformation was made. (The results of psb1c3 gel extraction was lower. So we performed only the first 7 gene ligation.) Creating “Plasmid pCAG”
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
Colony PCR of “pSB1C3 – GBlocks” (25.07.2015)
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)
G-Blocks PCR / Gel Electrophoresis (25.07.2015)
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “pSB1C3- GBlocks” (26.07.2015)
Result: negative Digestion of “pTEToff” (26.07.2015)
37˚C 1h Result: Bands were at the expected section. Gel purification was made. GEL GÖRÜNTÜSÜ
Creating “Plasmid pCAG” – Continue (27.07.2015)
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)“
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “pSB1C3 – Gblocks” (27.07.2015)
Sonuc: Only 16-9 is positive. GEL GÖRÜNTÜLERI
Colony PCR of “pSB1C3 – Gblocks” (28.07.2015)
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. tab2wiki
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Total: 20 ul 37˚C overnight Ligation will be made.
Ligation of “GBlocks (4,5,6,7,8,10)” and “PET45 (X+B)”
Room Temperature 2h
Room Temperature 2h
Creating of “pTRE – mLacI miRNA-BS” (27.07.2015)
Gel purification was made. GEL GÖRÜNTÜSÜ
Creating of “pTRE – mLacI miRBS” (27.07.2015)
Room Temperature 2h Transformation was made with TOP10.
Creating of “pTEToff – miRBS” (27.07.2015)
Creating of “pTEToff – miRNA Switch miR BS” (28.07.2015)
Room Tempretare 2h Transformation was made.
Creating of “pCAG”
Mini Prep of “Colony PCR 16-9”
Mini Prep of “pTEToff”
Cut Check of “HNS toehold and Trigger RNA” (30.07.2015)
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
Gel extraction 8-9
Colony PCR of „pSB1C3 – Gblocks“
Result: negative GEL GÖRÜNTÜLERI
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
#
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
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
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
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
#
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
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’
#
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
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
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
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
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
#
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
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
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
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
#
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
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
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)
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
#
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
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
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
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
#
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
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
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
#
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
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
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
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
Cycling
95˚C
95˚C
56˚C
72˚C
72˚C
Cycle
Time
5’
30’’
30’’
1.5’
5’
35x
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
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
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
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
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
#
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
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
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
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
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
#
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
#
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
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
#
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
#
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
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
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!
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 |
Colony PCR of „pSB1C3 – Gblocks“ (03.08.2015)
Result: negative
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