Wednesday 10/14/15

DNA Extraction of Oly Broodstock

  • SS1_1
    SS1_2 (not tan)
    SS1_3
    SS1_4
    SS1_5
    SS1_6
    SS1_7
    SS1_8
    SS1_9
    SS1_10
    SS1_11
    SS1_12
    SS1_13
    SS1_14
    SS1_15
    SS1_16
    SS1_17
    SS1_18
    SS1_19
    SS1_20
    SS3_1
    SS3_2
    SS3_5
    SS3_6
  • Found 2 tubes labelled SS1_2. Marked the unextracted one with a tan sticker
  • Could not initially find SS3_3 or SS3_4 but found later and reorganized box
  • Left in 60degC water bath for 3 hours
  • Eluted 50 uL then 100 uL

Primers

  • Received the rest of the 2b-RAD primers and resuspended them to 100 uM.
    • BC11
    • BC12
    • HT3
    • HT4
    • HT5
    • HT6
    • HT7
    • HT8
  • Made new 1 uM aliquots of BC2-10 and HT1
    • 1 uL stock + 99 uL NFW
  • Made new 10 uM  aliquots of Lib 1 and Lib 2
    • 2.5 uL stock + 22.5 uL NFW

PCR for 2b-RAD MiSeq run

Used 10 samples from 9/28/15 digestion and ligation. Numbers in parentheses are so I know which tube from the ligation each sample is in.

  1. SS2_6 (5)
  2. SS2_7 (6)
  3. SS2_8 (7)
  4. HC1_5 (8)
  5. HC1_6 (9)
  6. HC1_7 (10)
  7. HC1_8 (11)
  8. NF1_4 (14)
  9. NF1_5 (15)
  10. NF1_6 (16)

Master Mix

1x
10.5x
NFW
23 uL
241.5
10 mM (each) dNTPS
2
21
10 uM ILL-Lib1
2
21
10 uM ILL-Lib2
2
21
5X Q5 buffer
20
210
Q5 Taq polymerase
1
10.5
Added 50 uL of MM to 40 uL of ligation product. Added 5 uL of 1uM BC and HT to each sample, using BC1-10 and HT1.
Run out gel of 2b-RAD PCR product
This time, set up a medium gel for 8 of the samples and a small gel for the other 2. Made a 2% TAE gel, and after running it at 110V for 1 hour let the gels soak in water with 6 uL of EtBr to increase staining. PCR used 19 cycles.

Gel of 2b-RAD PCR product

Gel of 2b-RAD PCR product

20151015_124156

I think I should make sure to mix in the EtBr when doing the post staining as that is likely why there are bright white sections on the gels. It was still much easier to see the bands though. Cut out the bands and left them in 1.5 mL tubes in 4degC for gel extraction on Friday.

Advertisements

One thought on “Wednesday 10/14/15

  1. Pingback: Friday 10/16/15 | Getting My Genes Wet

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out / Change )

Twitter picture

You are commenting using your Twitter account. Log Out / Change )

Facebook photo

You are commenting using your Facebook account. Log Out / Change )

Google+ photo

You are commenting using your Google+ account. Log Out / Change )

Connecting to %s