NOTEBOOK
December
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January
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February
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March
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April
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May
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June
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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. 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..
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
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..