Joyce
Calculate with the ligation calculator how much of the insert is needed for a vector: insert ratio of 1:6
Digestion:
3μl RBS-GFP-DT
1μl EcoRI
1μl XbaI
2μl FD buffer
13μl MQ water
insert:
4μl insert
1μl EcoRI
1μl SpeI
2μl FD buffer
12μl MQ water
Digestion one reaction:
1μl pSB1C3 (50 ng)
0.5μl insert
0.5μl EcoRI
0.5μl PstI
2μl FD buffer
15.5μl MQ water
Digest for 1h at 37 degrees
DNA clean up with High Pure PCR Purification Kit
Ligation:
For the 1 digestion reaction: elute in the purification step in 17μl
Add: 2μl T4 DNA ligase buffer and 1μl T4 DNA ligase
Ligation promotors upstream RBS-GFP-DT
2μl T4 DNA ligase buffer
1μl T4 DNA ligase
8.5μl vector
6μl insert
2.5μl MQ water
Transformation
Add to 40μl competent cells 10μl ligation mixture
Incubate for 30 min on ice
Heat shock: 45s at 42 degrees
Incubate cells on ice for 2 min
Add 1ml LB medium+ 25mM glucose
Incubate cells for 1h/1.5h at 37 degrees
Spin the cells down, resuspend them in 100μl LB medium and plate 90μl and 10μl out on the plates
Put the plates in the stove at 37 degrees overnight
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