User:Anthony Salvagno/Notebook/Research/2009/09/02/Sap Cap AGC suspension and annealing

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For protocol see here: Resuspension of T, B, and HP oligos

Resuspension

pmols:17718

  1. add 177uL of .1x TE
  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:
    • 820ng/uL - with a MW of 8929g/mol this gives me a concentration of 92uM which is close enough! SJK 01:06, 3 September 2009 (EDT)
      01:06, 3 September 2009 (EDT)
      Yup, that's definitely about as close as I usually got!
    • again, we want 100uM

Annealing

The idea is to heat and quick chill. So I am going to follow the protocol on above mentioned page: 5min at 100C and 5min on ice.

I really don't want to make a google doc for this one reaction so I'll just detail below:

  1. made annealing buffer from 10x TE and 3.8M NaCl:
    • 1uL of 3.8M NaCl in 75uL 10x TE (that is my logic to get 50mM NaCl + TE)
  2. combined
    • 6uL sapcap
    • 10uL 10x anneal buff
    • 84uL ddH2O
  3. heat at 95C for 5 min
  4. cool on ice for 5 min
  5. store in box #3 as Sap Cap

Tomorrow is ligation day.

Basing off the above results, I put 5ug DNA in the above reaction. That will give me 50ng/uL in solution for a total of 5.6uM. I don't know if making it double stranded will decrease the molarity or what. I could do a nanodrop, but I don't wanna.