Team:PKU Beijing/Notebook/AND Gate 1/Input/LacI TetR 5

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Delete LVA tail from reverse tetR-tetP-GFP

Resource

Reverse tetR-tetP-GFP: from Lin Min, plasmid. Renamed as TPG
Reverse tetR-tetP*2, (not very confirm): from Lin Min, plasmid, Renamed as TP1, TP2
Vector: a plasmid with Kan resistant. From Lin Min
Primer:
Delete LVA primer, with complement to 20 last bps of tetR coding sequence and a TAA+Xba1 tail. And it can be use with one of standard primers (Rev), to amplify any sequence between the end of tetR and standard suffix.
5’-GCTCTAGATTAGGACCCACTTTCACATTTAA-3’
Designed by myself.

2009.8.11

PCR: (helped by He Siheng)

System20 uL
pfu enzyme1ul
primer1uL eachdelete LVA primer and standard primer reverse one
Buffer2 uL
water10uL
template TPG1uL
dNTP4uL

Gel electrophoresis:
Products of PCR;
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min
lane1: Marker;
lane2: product;
PKU 20090811 Shuke Wu 1.JPG
Obviously, PCR is failed!!!
Repeat!!

PCR: (repeat)

System20 uL
pfu enzyme1ul
primer1uL eachdelete LVA primer and standard primer reverse one
Buffer2 uL
water10uL
template1uLTP1, TP2, TPG
dNTP4uL

extending time 5 min;

2009.8.12

Gel electrophoresis:
Products of PCR;
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min
PKU 20090812 Shuke Wu 1.JPG
Lane1: TP1,
Lane2: TP2,
Lane3: TP1 (use another standard primer for one)
Lane4: Marker;
Lane5: negative control;
Lane6: TPG
The result is very strange:
TP1 and TP2 should be about 1kb, but there is not!
TPG should be about 2kB, but another 0.8k also very strong.

DNA Gel purification:
TPG (2kb) insert

Double digest:
TPG insert:

Xba11uL
Pst11uL
DNA16uL
Buffer2uL

Vector:

Xba11uL
Pst11uL
plasmid4uL
Buffer2uL
water12 uL

37 ℃ 4 hour

Gel electrophoresis:
Products of digestion of vector
marker: 100bp 250bp 500bp 750bp 1kb 2kb 3kb 5kb
loading buffer and DNA dye: 6×
voltage and time: 60V 5min; 120V 15min
PKU 20090812 Shuke Wu 2.JPG
The vector is about 3kb.

DNA Gel purification:
Vector,

PCR product purification:
Products of digestion of insert (TPG)

DNA ligation:

System10uL
Insert6uL
vector2uL
buffer1uL
T4 DNA ligase1uL

16℃ 4 hour
Insert: TPG;
Vector

Transformation:
Products of ligation, competent cells 50uL each,
Smear to LB plate with Kan.

2009.8.13

The plate (delete LVA TPG) is very well: more than 100 clones
And many colonies are become green under the blue light, which means that the expression of tetR can not fully repressed the promoter tetP.
The second picture is for comparison with no GFP colonies.
PKU 20090813 Shuke Wu 1.JPG

PKU 20090813 Shuke Wu 2.JPG

2009.8.14

Plasmid mini prep:
Del-LVA-TPG1, 2, 3.

Result

I successfully deleted the LVA from the Reverse tetR-tetP-GFP. Yet, the result is not very promising, because the colonies became green on the plate, without inducing.
It needs more quantitative data, but it is obviously that this cloning does not work very well.

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