NOTEBOOK
December
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
January
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
February
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
March
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
April
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
May
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
June
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
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
Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description.. Some description..
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 | ???˚C | 72˚C | 72˚C | Cycle | |
| Time | 2’ | 10’’ | 30’’ | 1’ | 5’ | ???x |
| 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 | ???˚C | 72˚C | 72˚C | Cycle | |
| Time | 2’ | 10’’ | 30’’ | 1’ | 5’ | ???x |
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
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
6C??
GC??
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
August
Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance..
Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance..
Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..
Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..
September
Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance.. Background of acid resistance..
Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance.. Design of acid resistance..
Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..
Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance.. Results of acid resistance..