User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/03
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 Alien genes
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Juno2 probe preparation from P. roseola
Amplifying Juno2 from P. roseola
| Reagents | 1x | conc. |
| h2o | 29.75 | - |
| 10x | 5 | 10x |
| dye | 5 | 10x |
| dNTP | 1 | 10 mM |
| Forward | 2 | 10 uM |
| Reverse | 2 | 10 uM |
| Taq | 0.25 | 5 U/um |
| DNA | 5 | 50 nm/ul |
- Purified with Invitrogen kit. Yeild:
| 3m 95C (15s 94C; 3m 60C)x35 |
| 25.7 | ng/ul | 0.03852 | pm/ul |
 |
 |
Single stranded probe preparation
| h2o | 8.75 | - | |
| 10x | 5 | 10x | |
| dNTP | 1 | 10 mM | |
| Forward | 10 | 10 pm/ul | 50 pmol total |
| Taq | 0.5 | 5 U/um | |
| DNA | 25 | 0.03852 pm/ul | 1 pmol total |
| TOTAL | 50 |
| 3m 95C (10s 94C; 2m 60C)x60 |
- Second band should be single-stranded DNA. I hope. Frozen @ -20.
- This should give us an approx 1 μM solution of single-stranded probe, and can be used for hybridization without further purification.
Trying to get some long DNA segments from P.rapida
- got some large molecular weight DNA from Priya
- Calculations:
| ul gel | U@15min | U@overnight | U/ul agarACE | ul AgarACE |
| 1U/200ul | .025U/200ul | |||
| 17000 | 85 | 2.125 | 0.29 | 7.327586207 |
- Melted gel at 65C, then equilibrated at 42C for 10 min.
- Added 7.3 ul AgarACE at 7pm
