User:Alexander S Mikheyev/Notebook/rotifer alien genes/2009/11/03

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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
Gid 2009-11-03 17hr 46min.jpg
Dsdna091103.JPG

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

Gid 2009-11-04 10hr 15min.jpg

  • 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