precipitation
- 0.5 volume (125 ul) of 7.5M NH4OAc, mix,
- then add 2.5 volumes (950 ul) of room temperature 95% ethanol
- 1 hour @ -20C
- spin max speed 15 min
- wash with 200 ul cold 80% ethanol
- spin 5 min max
- dry, re-suspend in 15 ul water
- there is about 15 ul of gel left
- looks like crap on the nanodrop (huge peak at 230)
PCR of ligated product
| DNA (2 μl)
|
| Phusion DNA polymerase (Finnzymes Oy) (25 μl)
|
| PCR primer 1.1 (1 μl)
|
| PCR primer 2.1 (1 μl)
|
| Water (22 μl)
|
| 30s 98C |
(10s 98C; 30s 65C; 30s 72C)x10 |
5m 72C
|
| 2 ul of the product on a gel |
after cleaning
|
 |
|
- 6 ng/ul (61 fmol/ul) ==> 180 ng total yield
- frozen at -20C
|
Alien genes
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