Kun:LabNotes/CpgSeq/2010-6-8: Difference between revisions
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m (Created page with '==Probe prep for the DMR220k set== Library name OLID Part # DMR220k_a 0284601 G4446A DMR220k_b 0284591 G4…') |
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Purify the amplicons with two Qiaquick columns, eluted with 40ul EB. | Purify the amplicons with two Qiaquick columns, eluted with 40ul EB. | ||
===Production PCR=== | ===Production PCR=== | ||
10nM 1st round amplicon 5ul | |||
2x Kapa SYBG qPCR Master Mix 50ul | |||
100uM pAP1V61U 0.4ul | |||
100uM AP2V6 0.4ul | |||
H2O 44ul | |||
=================================== | |||
100ul x 16 | |||
95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 14 -> 60C 2min -> 15C hold. | |||
Ethanol precipitation. | |||
===Digestion and purification=== | ===Digestion and purification=== |
Revision as of 21:32, 9 June 2010
Probe prep for the DMR220k set
Library name OLID Part # DMR220k_a 0284601 G4446A DMR220k_b 0284591 G4446A DMR220k_c 0284581 G4446A DMR220k_d 0284571 G4446A
- Resuspend the oligos in each tube with 500ul nuclease-free H2O.
Expansion PCR
x 4 DMR220k_a (20nM) 1ul DMR220k_b (20nM) 1ul DMR220k_c (20nM) 1ul DMR220k_d (20nM) 1ul 2x iTaq master mix: 100ul 100uM AP1V6: 0.8ul 100uM AP2V6: 0.8ul H2O: 96ul 94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 16 -> 72C 3min -> 15C hold Purify the amplicons with two Qiaquick columns, eluted with 40ul EB.
Production PCR
10nM 1st round amplicon 5ul 2x Kapa SYBG qPCR Master Mix 50ul 100uM pAP1V61U 0.4ul 100uM AP2V6 0.4ul H2O 44ul =================================== 100ul x 16 95C 30sec -> (95C 3sec -> 55C 30sec-> 60C 20sec) x 14 -> 60C 2min -> 15C hold. Ethanol precipitation.