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.
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
- Top Oligo: 554ng/uL
- Bottom Oligo: 2258.5ng/uL
- Hairpin Oligo: 2442.6ng/uL
- Top: 45uM
- Bottom: 217uM
- HP: 84uM
based on molecular weight.
- 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.