Alice:LabNotes/2008-9-14

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extending probe length

PCR after circlehelper insertion:

H2O                                                40ul       
SYBR  green (50x)                                 0.4ul          
Forward primer AmpRV6.2_2(100uM)                  0.2ul          
Reverse primer AmpFV6.2_2(100uM)                  0.2ul
Taq master mix(2x)                                 50ul         
probe template (product after Exo I&III)           10ul

94 2min -> (94 30sec -> 55C 1min -> 72 45 sec)* 15 cycles -> 72C 5min -> 15C hold

T7 exonuclease digestion:

Add 2ul of T7 exonuclease to the PCR product
25C 2hr -> 4C
Purify with Qiaquick columns, and measure the concentration using Nanodrop to determine the amount of spacer needed.
Nanodrop result: 

22K probe preparation using Ribo V6

Add 40ul of KOH to the remaining product after DpnII, incubate at 94C for 1hr.