Alice:LabNotes/2008-11-4

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diagnosing spacer annealing and insertion efficiency

For the product and procedure from previous steps, please follow this link (10/14/08 LabNote):  [[1]]
Add 1ul of spacer to 99ul of ddH2O to dilute them to 100nM
the final concentration of spacer is 20nM, so 20nM x 50ul =  100nM x amount needed(ul)           
Add 10ul of each primer, denature at 95C for 5min, and then anneal at 65C for 20min.

spacer insertion:
Repeat-reaction system 2:
template (1) 680nM                  1.5 ul 
spacer mix (100nM each)              20 ul
10x Ampligase Buffer                  5 ul
Ampligase(5U/ul)                      1 ul
ddH2O                              15.6 ul
------------------------------------------
total                                50 ul