User:Anthony Salvagno/Notebook/Research/2009/04/27/Oligo Annealing

From OpenWetWare
< User:Anthony Salvagno‎ | Notebook‎ | Research‎ | 2009‎ | 04‎ | 27
Jump to: navigation, search

So last week sometime we got the oligos we ordered. One is the top oligo for the SapI adapter, and the other is a hairpin adapter with a NotI site. I need to resuspend the DNA and then make stocks.

Resuspension

I want 100uM stocks (100pmol/uL). I started with:

  • Top adapter: 11504pmol
  • Hairpin adapter: 27590pmol
  1. So I need to mix:
    • 115uL 0.1x TE into TA
    • 276uL 0.1x TE into HP
  2. After addition of liquid I vortex the solution for about 1 min.
  3. Let the stocks sit on ice for 60'
  4. Vortex for 1min again.
  5. Take nanodrop reading

Nanodrop results:

  • Top Oligo: 554ng/uL
  • Bottom Oligo: 2258.5ng/uL
  • Hairpin Oligo: 2442.6ng/uL
SJK 00:33, 28 April 2009 (EDT)
00:33, 28 April 2009 (EDT)
These resuspended volumes seem more off than I would expect based on my prior experiences. I wonder whether the nanodrop is more inaccurate? This reminds me that the best way to make sure you have equimolar portions of top & bottom is to titrate one against the other and run native PAGE. You look for the lane with the most duplex versus free oligos. I think we should buy some precast gels for this. Or pour them. SYBR Green or Gold (can't remember which) used to be a good dye for this. Radioactivity is even better, but probably not worth it.

This yields:

  • Top: 45uM
  • Bottom: 217uM
  • HP: 84uM

based on molecular weight.

Annealing

{{#widget:Google Spreadsheet |key=rJWJM5BtAYjQc9JobkG4w-w|width=500|height=300}}

  1. Mix solutions to above specifications.
  2. Let both tubes sit for 5min at 100C.
  3. To cool:
    • Tube HP (hairpin) must sit in ice bath for 5min
    • Tube T+B (Top and Bottom oligos) must sit in block and cool to room temp.