User:Anthony Salvagno/Notebook/Research/2009/04/27/Oligo Annealing
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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
- So I need to mix:
- 115uL 0.1x TE into TA
- 276uL 0.1x TE into HP
- After addition of liquid I vortex the solution for about 1 min.
- Let the stocks sit on ice for 60'
- Vortex for 1min again.
- 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)
![](https://s3-us-west-2.amazonaws.com/oww-files-public/e/ed/SJK_Comment_Heading.png)
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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- Mix solutions to above specifications.
- Let both tubes sit for 5min at 100C.
- 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.