- Trying to single rotifer-extractions using fusion primers designed for population genetics
PCR conditions
| Reaction mixture
|
conc.
|
1x
|
76
|
| water |
|
5.75 |
437
|
| TopTaq buffer |
10x |
1 |
76
|
| dNTP mix |
10 mM each |
0.2 |
15.2
|
| Primer mix |
5 uM each |
1 |
|
| TopTaq (QIAGEN) |
5U/ul |
0.05 |
3.8
|
| template |
instagene |
2 |
|
|
|
7 |
|
|
|
- 3 min @95C, (5s 95C, 1 min 60C)x35 on Takara machine
- using MIDs 1 through 12
- rotifers from Gushikawa 01
- each placed with 5-10 ul water into a PCR tube. Then 30ul instagene were added and incubated 30min at 56, 30min at 95C, vortexing occasionally
Results
- Pretty much blank gel. There was some weak amplification in the last sample, but it looks like the extractions have failed.
- Need to optimize single-rotifer extraction
|
Alien genes
Main project page
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