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

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(2009.8.13)
 
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[[Team:PKU_Beijing/Notebook|Notebook]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1|AND Gate 1]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1/Input|Input]] > [[Team:PKU_Beijing/Notebook/AND_Gate_1/Input/LacI|IPTG Sensor ]]
==='''2009.7.30'''===
==='''2009.7.30'''===

Latest revision as of 18:46, 21 October 2009

 
Notebook > AND Gate 1 > Input > IPTG Sensor

2009.7.30

Measure the concentrations of Ligation products of reverse lacI + promoter

SampleConcentration(×50µg/mL)
11.6536
22.4225
32.7404
42.8690
52.4858
62.5618

Enzyme digestion to test the ligation product

Plasmid8µl
EcoRI1.5 µl
PstI1.5µl
10×H Buffer2µl
ddH2O7µl
20µl

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

SampleConcentration(×50µg/mL)
Promoter2.5671
Reverse lacI3.9938

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

Plasmid(promoter)10µlPlasmid(reverse lacI)10µl
EcoRI1.5 µlEcoRI1.5 µl
XbaI1.5µlSpeI1.5µl
10×M Buffer2µl10×H Buffer2µl
ddH2O5µlddH2O5µl
20µl20µl

2009.7.31

Using CIAP to recycle the vector

CIAP0.5µl
Buffer2.5 µl
Digestion product20µl
ddH2O2µl
25µl

37 centigrade, incubate for 30 min

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

Buffer1µl
Ligase1 µl
Vector(promoter)2µl
Fragment(reverse lacI)6µl
10 µl

Transform ligation product into competent cells
Procedure:

Ice bath30 min
Heat shock90 s
Ice bath2-3 min
Add 100 µl non-resistant LB
Shaker, 37 centigrade45 min
Plate on Amp plate

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

SampleConcentration(×50µg/mL)
113.7421
123.4852
171.9614
212.8062
255.3108
313.4985

Double digestion the plasmid

Plasmid5µl
EcoRI1.5 µl
PstI1.5µl
10×H Buffer2µl
ddH2O10µl
20µl

12:30
bath in 37 centigrade water
PKU 20090805 Siheng He 1.JPG

2009.8.6

Add SpeI and continue digestion
PKU 20090806 Siheng He 1.JPG

2009.8.7

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

SampleConcentration(×50µg/mL)
120.4524
170.5731

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

2009.8.8

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

2×superMix5µl
Universal Primer (Formard)0.25 µl
Universal Primer (Reverse)0.25µl
ddH2O4.5µl
10µl

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

SampleConcentration(×50µg/mL)
21.3526
52.9598
Plasmid 12-2supD6µl
EcoRI1.5 µl
PstI1.5µl
10×H Buffer2µl
ddH2O9µl
20µl

2009.8.10

Enzyme digest plasmid 5 to get the insert: reverse lacI+promoter
PKU 20090810 Siheng He 1.JPG

Digestion confirmation
PKU 20090810 Siheng He 2.JPG

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
PKU 20090811 Siheng He 1.JPG

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.

SampleConcentration(×50µg/mL)
5.71.4882
5.81.9782
5.91.5369

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.
PKU 20090812 Siheng He 1.JPG
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.
PKU 20090812 Siheng He 2.JPG

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.
PKU 20090813 Siheng He 2.JPG

2009.8.16

Double enzyme digestion: lacI+promoter+GFP delete LVA plasmid
PKU 20090816 Siheng He 1.JPG

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