User:Anthony Salvagno/Notebook/Research/2009/09/02/Sap Cap AGC suspension and annealing
For protocol see here: Resuspension of T, B, and HP oligos
- add 177uL of .1x TE
- 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:
- 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)
- again, we want 100uM
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:
- 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)
- 6uL sapcap
- 10uL 10x anneal buff
- 84uL ddH2O
- heat at 95C for 5 min
- cool on ice for 5 min
- 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.