2009.9.4
Colony PCR results
H1~5: sal-supD-lacI-T7ptag, Amp plate
S1~5: sal-supD, Amp plate (provided by WSK)
Miniprep S1, S2, S4
Double digestion (EcoRI+PstI): S1, S2, S4
40 uL S1(sal-supD, A+K+) is digested using EcoRI and PstI
S2(sal-supD, A+K+) is digested using EcoRI
| Plasmid | 3µl
|
| EcoRI | 1 µl
|
| 10×H Buffer | 1µl
|
| ddH2O | 5µl
|
| ∑ | 10µl
|
Prepare salicylate promoter insert and supD vctor
| Sal-1M plasmid | 5µl
|
| XbaI | 1 µl
|
| PstI | 1µl
|
| 10×M Buffer | 2µl
|
| ddH2O | 11µl
|
| ∑ | 20µl
|
| supD+terminator plasmid | 5µl
|
| SpeI | 1 µl
|
| PstI | 1µl
|
| 10×H Buffer | 2µl
|
| ddH2O | 11µl
|
| ∑ | 20µl
|
2009.9.5
Single digestion using EcoRI: supD+terminator plasmid
Double digestion using EcoRI and PstI: supD+terminator plasmid
Electrophoresis
Lane1: plasmid 1
Lane2: plasmid 1 single digestion
Lane3: plasmid 1 double digestion
Lane4: plasmid 2
Lane5: plasmid 2 single digestion
Lane6: plasmid 2 double digestion
13:00
double digestion using EcoRI and XbaI: supD+terminator plamid
double digestion using EcoRI and SpeI: sal-1M plamid
16:00
double digestion using EcoRI and PstI (NEB enzyme): supD plasmid
21:00
electrophoresis to identify the results of digestion
Lane 1: plasmid
Lane 2: plasmid double digestion (EP)
There is a band whose length is about 300 bp and it demonstrates that the supD plasmid is right.
Recycle the Sal insert from gel. The insert is about 1.3 kb in length
DNA product purification: digested supD plasmid
22:30
ligation: sal (insert)+supD(vector), T4 DNA ligase (NEB), reaction under room temperature
Transformation
2009.9.6
01:00
plate (one is A+K+, the other is A+)
Pick colonies from A+K+ plate and PCR
No.4 and No.5 are still transparent after 11 hours, MiniPrep plasmid from No.1~3 tube
2009.9.7
3:30
No.2 plasmid is double digested with EcoRI and SpeI, and the insert is given to ZHQ
No.2 plasmid is double digested with EcoRI and PstI, and the insert will be linked to another backbone (1-7G)
12:30
electrophoresis
13:00
gel extraction: sal+supD insert
15:10
ligation: sal+supD (insert) and 1-7G (vector)
16:00
ligation
17:20
plate Amp plate and Kan plate. Amp plate is check if there is pollution of previous plasmid
2009.9.8
9:00
there is no colony on the Amp plate, shown that there is no pollution
Pick 5 colonies from Kan plate, PCR and shake in the incubator
20:30
double digestion to get 9 kinds of RBS+T7ptag+lacI: 1-1J, 1-5N, 1-1H, 1-2M, 1-2K, 1-5J, 1-2G, 1-1N, 1-2I
| Plasmid | 3µl
|
| XbaI | 1 µl
|
| PstI | 1 µl
|
| Buffer | 2µl
|
| BSA | 0.2 µl
|
22:15
MiniPrep the 5 tubes and numbered SS(1)~SS(5) 1-7G
3 µl plasmid is double digested with SpeI and PstI for recycle
3 µl plasmid is double digested with SpeI and PstI for identification
2009.9.9
00:30
gel extraction: 9×RBS+lacI+T7ptag, 9 insert in total
DNA product recycle: sal-supD 1-7G (digested with SpeI and PstI) as vector
Sal-supD 1-7G double digestion identification results. The insert is about 1.4 kb in length and can hardly been seen on the gel. Only backbone can be seen in the figure.
4:00
ligation: 9×RBS+lacI+T7ptag (insert) and sal-supD plasmid (vector)
11:30
transform ligation products into JM109
13:00
spread Kan plate
2009.9.10
00:00
only 1-5J, 1-2K, 1-5N, 1-1N, 1-1H plates have colonies. Colony PCR
Double digestion with EcoRI and PstI: sal-supD plasmid, overnight
Double digestion with SpeI and PstI: sal-supD plasmid, overnight
10:00
electrophoresis: sal-supD plasmid (EP digestion)
The band located at 1.3 kb is more clear than yesterday
16:00
double digestion: 9×RBS+lacI+T7ptag plasmid
21:30
electrophoresis, gel extract the insert
23:00
ligation: sal-supD + lacI-T7ptag
24:00
transformation
2009.9.11
01:35
plate
12:00
colony PCR
16:00
The PCR results are all negative
20:00
pick 5 colonies separately from 5J and 2G plate and grow in incubator
2009.9.12
13:00
MiniPrep 5J 1~5, 2G 1~5
14:00
double digestion with EcoRI and PstI to identify: 5J 1~5, 2G 1~5 plasmid
2009.9.22
16:00
revive the 2G and 5J bacteria cell
20:00
induce 2G and 5J with IPTG and salicylate
22:00
use to measure the fluorescence intensity of induced cells
2009.9.23
12:00
1:50 revive the bacteria containing T7 promoter+GFP plasmid to OD600nm≈0.4
18:00
prepare competent cell using the revived cell
18:30
transformation
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