PCR of ligated product
| Reagents
|
1x
|
conc.
|
| h2o |
30 |
-
|
| 10x |
5 |
10x
|
| dNTP |
2.5 |
10 mM
|
| Linker_F |
4 |
10 uM
|
| Taq |
0.4 |
5 U/um
|
| DNA |
8 |
4.4ng (~.45 fmol)/ul
|
| TOTAL |
50 |
Cocktail per vial
|
| 3m 93C (15s 93C; 15m 65C)x15
|
| 2 ul PCR product left lane
|
|
- add 1ml of 1M NaCl to a 6 well dish filled with rotifers, they curl up
- each well is about 10ml, resulting in a 100mM NaCl solution
- spin at 1000g for 5 minutes
- put into 1.5 ml tube, spin again
- wash with small amount of water and go to extraction kit (quiagen micro)
- yield 7ug for vaga and 5ug for ricciae
| 60 ng A. ricciae DNA, far right lane
|
|
|
|
Alien genes
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