Alice:LabNotes/2008-10-6

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PCR amplification of cosmic probe

PCR reaction system                                      x48              
H2O                                       50ul          2400ul              
SYBR  green (50x)                        0.4ul          19.2ul
primer mix (100uM)                       0.2ul          9.6ul                 
Taq master mix(2x)                        50ul          2400ul 
probe template (cosmic)                 0.05ul          2.4ul
 
94c 2min -> 94c 30sec -> 60C 2min -> 72C 1min-> Plate read -> (94c 30sec -> 60C 2min -> 72C 1min)x 20 cycles -> 72C 5min ->
15C hold

Purify the product using ethanol purification.

T7 exonuclease digestion

1. The tubes were purified using ethanol purification, and diluted in 100ul H2O for every 24 reactions. 
2. Add ratio of 0.5ul/tube amount of T7, which is 12ul of T7 and 11ul of NEBuffer #4 to each tube(24 reactions).
3. 25C for 2 hrs.
4. Qiaquick column purification, elute in 30ul of H2O.
5. Measure with Nanodrop.
Nanodrop result: 

Circularization reaction

the concentration of the template from last step is ng/ul, so it is equivalent to

reaction system 1: 1:1 ratio of template:circleHelper

reaction system 1: template ( nM) 2ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 0.5ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 14ul

94C 2 min -> 55C 20 min

add 1.5ul Exo I &III + 10ul of product from reaction system 1 37C 2hr -> 95 10min -> 4C Purify using Minelute columns

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: 19.8ng/ul *30ul