Jie:LabNotes/CpgSeq/2008-8-1
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Insertion of spacer in cpg30k_v2.2
PCR amplification of cpg30k_V2.2
reaction system x48 H2O 80.7ul 3873.6ul 10x buffer 10ul 480ul MgCl2(25mM) 6ul 288ul dNTP(10mM each, NEB) 2ul 96ul Ap1V4IU (100uM) 0.2ul 9.6ul Ap2V4 (100uM) 0.2ul 9.6ul SYBR reen (50x) 0.4ul 19.2ul Jumpstart Taq 0.6ul 28.8ul template(cpg30KV2.1,20uM) 0.05ul 2.4ul Total 100ul 4800ul Reaction program: 94c 2min -> 94c 30sec -> 60C 1.5min -> 72C 1min-> Plate read ->
(94c 30sec -> 60C 1.5min -> 72C 1min)x 22ycles -> 72C 5min -> 15C hold. Ethanol precipitation. Add 125ul ddH2O to amplicons from 24 PCR tubes. Add 15ul 10x Lambda Exonucleus buffer and 8ul lambda Exonucleus, 37C 3h and 90C 10mins. Quiquick column purification. Elute in 60ul ddH2O. yield: ssDNA: 157ng/ul x 60ul.
annealing of cirlehelper
The probe:circlehelper is expected to be 1:1. Probe concentration is: 157 ng/ul /(100x330) = 454 nM. 6ul of probe is 28545 x 10^-15 mol. The volume of CircleHelper(100uM) needed is 0.28545ul.
Annealing system is setup is as follows: template 6ul 3 3 10x Ampligase Buffer 3ul 3 3 Circlehelper(100uM) 0.3ul 0.15 0.1 dNTP(10mM) 0.5ul 0.5 Stoffel fragment(10U/ul) 0.5ul 0.5 Ampligase 1ul 1 ddH2O 18.7ul 21.85
94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C . check product on TBU gel.
File:20080801 after circlehelper ligation.jpg
recalculate: Probe concentration is: 157 ng/ul /(150x330) = 317 nM. 3ul of probe is 9515 x 10^-15 mol. reset the annealing system is setup is as follows: template 3ul 10x Ampligase Buffer 3ul Circlehelper(10uM) 1ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase 1ul ddH2O 21ul
94C 3min -> 5 cycles X (94C 30sec -> 60C 10mins) -> 4C .
Pool two sets and do PAGE purification of digested product. Quantification: 2ng/ul(40nM)
anneal with AluI oligo and digestion with AluI
1. Add 1ul NEB buffer 1, 2 uL of 100 uM RE-AluI-V6 guide oligo in 10ul of template from previous steps(ratio is 500:1). Denature at 95C in PCR machine for 10 min. Turn off the machine and wait for 20 min. 2. Add 1 uL of AluI restriction enzyme to reaction, incubate at 37C for about 2 hours. 3. Purify the reaction with Minelute columns. Elute in 10ul ddH2O.
PCR amplification of spacer_107bp
reaction system x96 H2O 80.7ul 7747.2ul 10x buffer 10ul 960ul MgCl2(25mM) 6ul 576ul dNTP(10mM each, NEB) 2ul 192ul spacer_100bp_F(100uM) 0.2ul 19.2ul spacer_100bp_R(100uM) 0.2ul 19.2ul 50x SYBG I 0.4ul 38.4ul template(E.coli clone) 0.1ul 9.6ul Jumpstart Taq 0.6ul 57.6ul Total 100ul
94C 3min -> 8 cycles of (94C 45sec -> 60C 45sec -> 72C 45sec) -> 20 cycles of (94C 30sec -> 68C 45sec -> 72C 30sec)-> 72C 3min -> 4C
Ethanol precipitation. Add 125ul ddH2O to amplicons from 24 PCR tubes. For each 24 PCR amplicons, add 15ul 10x Lambda Exonucleus buffer and 8ul lambda Exonucleus, 37C 3h and 90C 10mins. Quiquick column purification. Elute in 30ul ddH2O. yield: ng/ul x 30ul.