NOTEBOOK
December
(9-13 December)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.
(22.12.2014)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.
(26.12.2014)Had our first meeting and our first topic was: sponsorship.
January
We had training classes at 8-9-10 January. Now it is time to learn what iGEM wants from us.
(10-23 January)We made researches about previous iGEM teams, wondering who did what.
(23-28 January)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
(01.02.2015)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.
(02.02.2015)We decided to attend some competitions due to the lack of sponsors. We attended ‘lllnesses with imagery’. Talented people is a must in a team.
(13.02.2015)Drawings were done and sent. We believe wewill be successful. We are still doing researches in full swing.
March
(05.03.2015)Today they filmed our school, and our laboratory so. We had so much fun while they were shooting us!
(11.03.2015)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.
(14.03.2015)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 :)
(17.03.2015)Lab-cleaning party! We don’t wanna be contaminated.
April
05.04.2015 We bid farewell to our teammate Furkan Beştepe. He went to USA.
10.04.2015 Happy birthday Gülnhial! By the way your birthday cake was yummy.
12.04.2015 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.
13.04.2015 We are still learnig about Toehold. It has a potential to be used.
27.04.2015 Fun at the lab.
30.04.2015 ATOMS Ladieas worked on a universal blood idea till the morning.
May
03.05.2015 We must hurry up to find a project. Camp time at Asya Thermal Hotel.
04.05.2015 Why there isn’t anyone in the lab?
06.05.2015 Finally found our project idea! ULCER AND CANCER
09.05.2015 Perfecting our cancer switch system.
15.05.2015 Presented our project to the dean Mehmet Gündüz and the whole genetic department of our faculty! He treated us with Turkish tea and snacks.
24.05.2015 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.
GEL GÖRÜNTÜSÜ
(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.
GEL GÖRÜNTÜSÜ
July
01.07.2015 Some preparetions were made for experiments.
05.07.2015 Today is the first day of shooting. We just learned picking costumes isn’t as easy as we think.
06.07.2015 If there is a film, then there is After Effects work to do. Good luck with this, dear teammate Şahika.
08.07.2015 We made a presentation to the pre-med students which came from USA for intership and they loved our project.
21.07.2015 Drawing pictures for wiki began. Thus we discovered our teammate Kevser’s hidden talents.
22.07.2015 Our gene blocks has just arrived. Let the experiments begin! We are all ready now.
29.07.2015 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.
GEL GÖRÜNTÜSÜ
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 GEL GÖRÜNTÜSÜ
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
11.08.2015 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)
Sonuc: negative GEL GÖRÜNTÜSÜ
Colony PCR of „pSB1C3 – Gblocks #14“ (03.08.2015)
Sonuc: negative GEL GÖRÜNTÜSÜ
Digestion of “pSB1C3-RFP” (03.08.2015)
37˚C 2h Gel Electropheresis was made. Result: Bands were at the expected section. Gel purification was made. GEL GÖRÜNTÜSÜ
Ligation of “pSB1C3 – Gblock #14”
PCR of “G-Bloks via Q5 Polymerase” (04.08.2015)
Digestion of “pSB1C3-RFP” (04.08.2015)
37˚C 4h Result: Bands were at the expected section. GEL GÖRÜNTÜSÜ
Colony PCR of “11,12,13 pSB1C3 - GBlocks” (04.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Ligation and Transformation of “5,6,7,8,9 pSB1C3-GBlocks” (04.08.2015)
50ng DNA 1 vector:0.5 insert Transfomation with TOP10. Ligation of “GBlocks 11-12 pSB1C3” (05.08.2015)
Transformation was made.
Colony PCR of “5,6,7,8,9,T7 system – Gblocks” (05.08.2015)
GEL GÖRÜNTÜSÜ
Ligation of “GBlocks 2,5,6,7,8,9,10,12,13,15,17 pSB1C3-RFP” (06.08.2015)
Transformation was made.
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “5,6,7,9 – Gblocks” (06.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “7,8,10 – Gblocks” from Masterplate (08.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Digestion of “pET45” (09.08.2015)
37˚C 3.5h Result: Positive. Gel extraction was made. GEL GÖRÜNTÜSÜ
16˚C 2h Digestion of “pSB1C3 – HNS”
37˚C 3h
Colony PCR of “pTRE #13”
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “PET45-4,5,6,7,8,9,10” (10.08.2015)
Result: Bands were at the expected section. Liquid Culture was made. GEL GÖRÜNTÜLERI
Digestion of „pColA-KanR and „pSB1C3-Toehold“ (10.08.2015)
37˚C 2h pColA-KanR: 1.0 ul rSAP/37˚C 1h/-80˚C 20’’ heat shock GEL GÖRÜNTÜSÜ
Ligation of “pColA-KanR and pSB1C3-Toehold”
22˚C 1h Mini Prep of “pColA-3,4,5”
Result: The Band of pColA were at the expected section. 2000 pb (CUT CHECK GÖRÜNTÜSÜ)(12.08.2015)
Ligation of “#2 GBlock with pET45” (11.08.2015)
22˚C - 1’40’’ Transformation was made.
Mini Prep
Cut Check of “pET45 1,4,5,6,7,8,9,10 – BamHI/XhoI” (13.08.2015)
Dogru cikan koloniler BL21 bakterisine transformasyon edildi ve sivi kültür yapildi. Sonrasinda protein izolasyon ve WB yapilacak. GEL GÖRÜNTÜLERI
Colony PCR of “pTRE – 12,13 GBlocks” (14.08.2015)
Sonuc: negative GEL GÖRÜNTÜSÜ
Colony PCR of “pColA – Toehold GFP” (14.08.2015)
Result: 2-4, 2-8, 1-6 Bands were at the expected section. Digestion of “pSB1C3-RFP” (14.08.2015)
37˚C – 3.5h Gel extraction was made.
Ligation of “pSB1C3-GBlocks”
22˚C – 1.30h Transformation with Neb10-β. Colony PCR will be made.
Colony PCR of “GBlocks – 2,5,6,7,8,9” (14.08.2015)
GEL GÖRÜNTÜLERI
Digestion/Gel Electrophoresis/Gel Extraction and Purification of “pSB1C3” (16.08.2015)
37˚C 1h Colony PCR of “pSB1C3 7,10,12,13 and pET45 2” (14.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Result: negative GEL GÖRÜNTÜSÜ
Miniprep of “pTet-off DH5α /Top10/NEB5fα/NEB10β”
Colony PCR of “pET45 #2.8” (15.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “pSB1C3 17,6,8” (S. 118/15.08.2015)
Result: 6: Liquid Culture was made. 8: sequencing will be done 17: Colony PCR will be repeated. GEL GÖRÜNTÜSÜ
Colony PCR of “pColA blunt”
Result: negative GEL GÖRÜNTÜSÜ
PCR of “Q5 DNA Polymerase for pCAG” (19.08.2015)
Result: Bands were at the expected section. PCR clean up will be made. GEL GÖRÜNTÜSÜ
Digestion of “pCAG – Clean up”
37˚C overnight Digestion of “pSB1C3 – mLacI miR373-BS” (19.08.2015)
37˚C 2.30h Ligation of “pSB1C3 – mLacI miR373-BS with pTRE”
22˚C 1.30h Transformation will be made. Colony PCR of “#6,8” (20.08.2015)
Result: Positive. Liquid Culture was made. Cut Check of “#6,8”
37˚C 40’ Result: positive GEL GÖRÜNTÜSÜ
Colony PCR “pSB1C3 17,6,8 – pColA, Damp-Pex, pTetoff” (19.08.2015)
GEL GÖRÜNTÜLERI
Mini Prep of “pSB1C3-#17”
Digestion of “pSB1C3 T7-10 and pET45 #4-1, #5-3, #6-6, #8-15” (21.08.2015)
37˚C 3h 1ul rSAP / 37˚C 1h 22˚C 2h -80 ˚C 20’’ Heat Inaktivation Colony PCR of “pTRE - #13 mLacI-miR373 BS” (21.08.2015)
Results: negative GEL GÖRÜNTÜLERI
Mini Prep of “pSB1C3 #6-25,26”
Cut Check of “pSB1C3 #6-25,26”
37˚C 3h Result: negative Digestion of “pTRE #12”
37˚C 3h Result: negative GEL GÖRÜNTÜSÜ
Digestion of “Damp-Pex pSB1C3” (22.08.2015)
37˚C 3h Ligation of “Damp-Pex pColA”
22˚C 1.30h Transformation with NEB5α. Colony PCR of “Damp-Pex + pColA” (23.08.2015)
Result: #5-4, #5-9 Liquid Culture was made. GEL GÖRÜNTÜSÜ
Mini Prep of “pTetoff #14-4,5,8” (22.08.2015)
37˚C 2h Result: negative GEL GÖRÜNTÜSÜ
37˚C 30’’ Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “pSB1C3 – T7 #4,5,6,8” (23.08.2015)
GEL GÖRÜNTÜSÜ
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Digestion of “pTRE #12-2” (24.08.2015)
37˚C 2h Result: Bands were at the expected section. Gel Extraction was made. Mini Prep
Digestion of “TEV-Protease pET45/pSB1C3 T7/pColA (24.08.2015)
37˚C 3h Ligation of “pCAGop with pTRE #12-2” (24.08.2015)
Transformation was made. Cut Check “pSB1C3-T7 #4,5,6,8” (24.08.2015)
30’ digestion Colony PCR of “pTRE #13” (25.08.2015)
GEL GÖRÜNTÜSÜ + pTRE13 cut check görüntüsü
Colony PCR of “pCAG-DsRed” (25.08.2015)
Result: Bands weren’t at the expected section. GEL GÖRÜNTÜSÜ
Cut Check of „pSB1C3 – TlpB“ (25.05.2015)
7˚C 35’ The colonies will be made glycerol stock. GEL GÖRÜNTÜSÜ
Ligation of “Damp-Pex” (25.08.2015)
37˚C 3h 22˚C 1.5h Transformation was made.
Colony PCR of “pCAGop-DsRed” (26.08.2015)
Result: 2 colonie will be made liquid culture and streak plate. Ligation and transformation will be repeated. GEL GÖRÜNTÜSÜ
Mini Prep of “pTRE #13-3,5” (26.08.2015)
Colony PCR of “pColA – Damp-Pex, pSB1C3 – TEV
Colony PCR “pSB1C3 – TEV Protease”
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR “pSB1C3 – TEV Protease” – Repeat (27.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “pTRE – Luc #14” (27.08.2015)
Result: Bands weren’t at the expected section. GEL GÖRÜNTÜSÜ
Gel Extraction of “Damp-Pex”
Colony PCR of “pCAGop-DsRed #12” (27.08.2015)
Result: Negative. GEL GÖRÜNTÜSÜ
Ligation of “pCAGop-DsRed” (27.08.2015)
22˚C 1.30h Transformation was made. Colony PCR of „pSB1C3-T7 #5“ (27.08.2015)
Result: #1-1 Liquid Culture was made. GEL GÖRÜNTÜSÜ
Digestion of “pSB1C3-T7 TEV Protease (17. and 18. Colony)” (27.08.2015)
37˚C 3h 65˚C 20’’ Heat Inactivation Result: negative GEL GÖRÜNTÜSÜ
Digestion of “pColA” (27.08.2015)
37˚C 3h 1.2 ul rSAP 37˚C 1h 20’’ Heat Inactivation Colony PCR of “pCAGop-DsRed #12-2. Colony streak” (28.08.2015)
Result: Negative GEL GÖRÜNTÜSÜ
Colony PCR of „pSB1C3-T7 #5“ (28.08.2015)
Result: #5-31 Bands were at the expected section GEL GÖRÜNTÜSÜ
Cut Check of „pSB1C3-T7 #5-31“
GEL GÖRÜNTÜSÜ
Digestion of “pSB1C3 #9” (28.08.2015)
3h was digested Result: #9-5 Bands were at the expected section GEL GÖRÜNTÜSÜ
Ligation of “pColA #9”
2h was ligasted Mini Prep of “pCAG-DsRed #12” (28.08.2015)
Cut Check “pCAGop-DsRed #12”
Result: negative GEL GÖRÜNTÜSÜ
Cut Check “pCAGop-DsRed #12” - Repeat
Result: negative Colony PCR of „pTEToff and pTEToff N term“ (28.08.2015)
Result: positive GEL GÖRÜNTÜSÜ
Mini Prep of “pSB1C3-T7 TEV Protease” (28.09.2015)
Mini Prep of “pSB1C3-T7 TEV Protease” (29.09.2015)
Mini Prep of “pCAGop-DsRed #1-1” (30.08.2015)
Cut Check of “pCAGop-DsRed #1-1”
37˚C 3h Result: negative GEL GÖRÜNTÜSÜ
Digestion of “pSB1C3-T7 TEV Protease” (30.08.2015)
37˚C 3h Result: Bands were at the expected section. Gel extraction was made. GEL GÖRÜNTÜSÜ
Ligation of “pSB1C3-T7 TEV Protease”
22˚C 1.5h Transformation was made. Gel Extraction
Colony PCR of “pColA – TEV” (31.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Colony PCR of “pColA – TEV” – Repeat (31.08.2015)
Result: negative GEL GÖRÜNTÜSÜ
Digestion of “PsicA for pSB1C3 and invF-sicA for ColA” (31.08.2015)
37˚C 10h 82˚C 20’ Heat Inactivation
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
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
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
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
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
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
#
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
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
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
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
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 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
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
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
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
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
#
Sample ID
User name
Date and Time
Nucleic Acid Conc.
Unit
A260
A280
260/280
260/230
Sample Type
Factor
1
blank
biospec
8/9/2015 5:04:14 PM
-0.6
ng/ul
-0.011
0.005
3.41
0.68
DNA
50.00
2
pet45 x+b
biospec
8/9/2015 5:05:26 PM
106.9
ng/ul
2.138
-0.017
0.59
0.45
DNA
50.00
4
psb1c3 e+s
biospec
8/9/2015 5:06:20 PM
133.3
ng/ul
2.666
0.913
1.90
0.40
DNA
50.00
Ligation
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
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
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
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
Digestion
pColA-KanR (500 ng/ul)
pSB1C3-Toehold (500 ng/ul)
NotI-HF (Neb)
Cut Smart Buffer
ddH₂O
Total
1
11.6 ul
-
1.0 ul
2.0 ul
5.4 ul
20.0 ul
2
-
6.5 ul
1.0 ul
2.0 ul
10.5 ul
20.0 ul
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
#
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
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
#
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
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
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
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
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
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
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
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
#
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”
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
Colony PCR of “pET45 2”
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
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
#
Sample ID
User name
Date and Time
Nucleic Acid Conc.
Unit
A260
A280
260/280
260/230
Sample Type
Factor
1
blank
biospec
8/16/2015 8:54:05 PM
-0.2
ng/ul
-0.004
-0.017
0.25
0.12
DNA
50.00
2
psb1c3 13-15
biospec
8/16/2015 8:55:29 PM
125.2
ng/ul
2.505
1.339
1.87
2.01
DNA
50.00
3
psb1c3 15-19
biospec
8/16/2015 8:56:24 PM
93.5
ng/ul
1.871
0.999
1.87
1.94
DNA
50.00
4
psb1c3 7-16
biospec
8/16/2015 8:57:33 PM
149.4
ng/ul
2.988
1.575
1.90
2.14
DNA
50.00
5
psb1c3 10-16
biospec
8/16/2015 8:58:18 PM
138.0
ng/ul
2.760
1.469
1.88
2.13
DNA
50.00
6
psb1c3 10-17
biospec
8/16/2015 8:59:03 PM
185.5
ng/ul
3.710
1.994
1.86
1.86
DNA
50.00
7
psb1c3 7-11
biospec
8/16/2015 8:00:54 PM
73.8
ng/ul
1.475
0.843
1.75
1.08
DNA
50.00
8
psb1c3 13-14
biospec
8/16/2015 8:01:53 PM
178.7
ng/ul
3.575
1.922
1.86
1.87
DNA
50.00
9
psb1c3 7-20
biospec
8/16/2015 8:02:54 PM
73.6
ng/ul
1.472
0.774
1.90
2.05
DNA
50.00
10
pcola toe 2-4
biospec
8/16/2015 8:03:57 PM
40.1
ng/ul
0.801
0.441
1.82
1.13
DNA
50.00
11
pcola toe 2-8
biospec
8/16/2015 8:05:31 PM
48.7
ng/ul
0.974
0.503
1.94
1.61
DNA
50.00
12
pcola toe 1-6
biospec
8/16/2015 8:06:09 PM
54.9
ng/ul
1.098
0.570
1.93
1.62
DNA
50.00
13
pcola blunt 1
biospec
8/16/2015 8:06:52 PM
23.9
ng/ul
0.479
0.244
1.96
1.24
DNA
50.00
14
pcola blunt 6
biospec
8/16/2015 8:07:30 PM
31.1
ng/ul
0.623
0.314
1.98
1.67
DNA
50.00
Ligation
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
#
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
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
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
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
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
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
#
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
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
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
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
#
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
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
#
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 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
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
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
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
#
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
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
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
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
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
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
#
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
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
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
<section class="accordion">
<input id"august-week4" name="august-week4" type="checkbox" />
<label for="august-week4">Week 1, 24.08.-31.08.</label>
<article class="ac-small">
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
#
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
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
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
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
#
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
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
Cycling
95˚C
95˚C
56˚C
72˚C
72˚C
Cycle
Time
5’
30’’
30’’
1.5’
5’
35x
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
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
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
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
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
#
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 “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”
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
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
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
#
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
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
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
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
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
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
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
Colony PCR
MgCl₂
(NH₄)2SO₄
VF
VR
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
13.0 ul
3.2 ul
196.8 ul
325.0 ul
Cycling
95˚C
95˚C
56˚C
72˚C
72˚C
Cycle
Time
5’
30’’
30’’
1.5’
5’
35x
Cut Check
DNA
EcoRI (FD)
PstI (FD)
Fast Digest Buffer
ddH₂O
Total
1x
???????
1.0 ul
1.0 ul
2.0 ul
?????
20.0 ul
Digestion
DNA (500 ng/ul)
NotI
Cut Smart Buffer
ddH₂O
Total
1x
???????
1.0 ul
2.0 ul
?????
20.0 ul
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
#
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
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
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
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
#
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
#
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
#
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
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
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
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
#
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
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
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
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
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” (01.09.2015)
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” (01.09.2015)
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” (01.09.2015)
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.
Görüntü
Result: pColA Bands were at the expected section. Midi Prep Solution is better.
Colony PCR of “pCAGop-DsRed and pCMV-lacO” (02.09.2015)
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.
GEL GÖRÜNTÜSÜ
Colony PCR of “invF-sicA and PsicA” (02.09.2015)
Result: PsicA: 1, 2, 6, 8, 9, 10, 11 Liquid Culture will be made.
GEL GÖRÜNTÜSÜ
Digestion of “invF-sicA for pSB1C3 and ColA” (02.09.2015)
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” (03.08.2015)
# | 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” (03.08.2015)
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” (04.09.2015)
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.
GEL GÖRÜNTÜSÜ
Mini Prep of „pCMV-Lacop-DsRed and pCAGop-DsRed“ (05.09.2015)
# | 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.
GEL GÖRÜNTÜSÜ
July
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Cut Check of “invF and sicA” (07.09.2015)
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’
GEL GÖRÜNTÜSÜ
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 ul
|
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” (08.09.2015)
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
GEL GÖRÜNTÜSÜ
Digestion of “pColA / pSB1C3 #9 / pColA-Toehold #2-8 / pSB1C3 T7 #10-17 / #18” (10.11.2015)
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> <p>pColA-Toehold #2-8: 1ul rSAP / 37˚C 1h / 80˚C 20’
Result: pColA and #10 gel extraction will be made.
GEL GÖRÜNTÜLERI
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 |