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

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

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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.