Team:PKU Beijing/Notebook/AND Gate 1/Core/Siheng He

Notebook > AND Gate 1 > Core > Siheng He's Note

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

Prepare salicylate promoter insert and supD vctor

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

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