Team:PKU Beijing/Notebook/AND Gate 1/Input/LacI

Notebook > AND Gate 1 > Input > IPTG Sensor

2009.7.30
Measure the concentrations of Ligation products of reverse lacI + promoter

Enzyme digestion to test the ligation product

The result is wrong, so MiniPrep plasmids containing reverse lacI or promoter respectively and measure its concentration

Digest the above plasmids with EcoRI and XbaI or SpeI, 37 centigrade overnight

2009.7.31
Using CIAP to recycle the vector 37 centigrade, incubate for 30 min

Recycle the vector and fragment Ligation of reverse lacI fragment into vector containing the promoter

Transform ligation product into competent cells Procedure:

2009.8.1
9:00 Pick up the colony and shake in the incubator

2009.8.2
11:00 Pick up 21 colonies and identify the results by PCR It turns out that all the plasmids are self-ligation of the vector

21:00 ligate reverse lacI insert with lacI promoter again

2009.8.3
01:00 transformaiton

14:01 do the ligation again using the vector provided by WSK

20:00 transform the ligation product into DH5a

2009.8.4
Colony PCR Lane1-39: my sample Other lanes: WSK’s sample The number of the right-sized insert: 1,2,3,5,11,12,17,18,19,21,22,23,25,26,27,31,32,33,35,37

22:25 pick up the colonies 11,12,17,21,25,31,35 and shake in the 37 centigrade incubator Since the vector used for ligation this time is enzyme digested overnight without addition of BSA by WSK, the PCR results finally has the right insert band, about 1.5 kb.

2009.8.5
Miniprep plasmid with No 11,12,17,21,25,31 and measure their concentration

Double digestion the plasmid

12:30 bath in 37 centigrade water

2009.8.6
Add SpeI and continue digestion

2009.8.7
Electrophoresis and cut the lacI insert band for recycle, measure the recycle product concentration

15:30 Ligation of lacI+promoter (insert) with GFP or supD

2009.8.8
18:00 colony PCR: 12lacI-supD, 17lacI-supD

21:30 Electrophoresis to check results

23:00 ligate the insert (reverse lacI+promoter) with vector (containing supD)

2009.8.9
The plate 12lacI-GFP has one colony, colony PCR to check the result Miniprep 12-2supD and 12-5supD

2009.8.10
Enzyme digest plasmid 5 to get the insert: reverse lacI+promoter

Digestion confirmation

As shown in the figure, there are two bands, vector and insert respectively, it should be right Recycle the insert in the gel and ligate it with GFP vector, then transform into DH5a

2009.8.11
Pick up nine colonies from the plate and PCR to check the results

As shown in the figure, 5.1-5.6 are all achromatic, but electrophoresis results show that they are self-ligation. Shake colonies 5.7-5.9 which are green and shake them in the incubator, then MiniPrep plamid after 10 hours.

The results indicate that lacI with degradation tag LVA may not inhibit the expression of GFP Use synthesized primer and universal reverse primer to PCR tetR-GFP plasmid, electrophoresis and cut gel to recycle the insert.

2009.8.12
Double enzyme digestion 5.7~5.9 plasmid, electrophoresis. It seems that only 5.8 insert is right Use delete LVA primer (forward primer as control) to PCR plasmid 5, 5.7, 5.8. As shown in the figure, to my surprise, 5F also shows positive result. Gel recycle of 5R. Since elongation time is short for 5.7 and 5.8, both results cannot be judged.

2009.8.13
Use dLVA primer to PCR plamid 5(no GFP), enzyme digestion and gel recycle the lacI insert, then ligate it with GFP vector.

Use dLVA primer to PCR plasmid 5.7 and 5.7(contain GFP), gel recycle to get the insert.

2009.8.16
Double enzyme digestion: lacI+promoter+GFP delete LVA plasmid