|
Rxn
# |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
PCR
product |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
2 |
|
PCR
TOPO vector |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
|
Salt
solution |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
|
Up
to 5 ul with water |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
Use
1 ul vector per rxn. PCR product
may vary between 0.5 and 2 ul.
Incubate
5’ at RT.
Ice.
Add
2 ul 0.5 M BME to cells.
Add
2 ul lig rxn to cells.
Ice
30’.
42ø 30”.
Ice
2’.
Add
250 ul RT SOC.
Shake
30’ at 37ø for amp resistant strains.
Spread
100 ul on amp/X-gal plate.
Amp
50 ug/ml final.
X-gal
40 ug/ml final.