AlanFung:LabNotes/Capturing/2014-1-2

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Revision as of 18:26, 3 January 2014 by >Alan6017518 (→‎PCR)
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Helping Dheeraj to test Klentaq capture reactions

  • Dheeraj reached out to me saying he wants to start a qPCR reaction on the Klentaq capture reaction, he was working with Noi to test the protocol, but Noi is sick and I will be helping to perform the qPCR
  • They have 12 samples and we will be amplifying the captured samples with qPCR using the same protocol as it in Stoffol capture
  • 1-6 done in condition 16 hours incubation 4 hours polymerization
  • the second set we did the same 20 hours incubation and 20 hours polymerization

Protocol

PCR amplification to check if capture works

Components 1x rxn (ul) 12x rxn (ul) Final Conc.
Circularized DNA template 2.5 0  
KAPA SSYBR FAST MM 2X 12.5 150 1X
10uM AmpF6.4Sol 0.5 6 200nM
10uM AmpR6.3Ind23 0.5 6 200nM
H2O 10 120  
Total 25    
  • 98C 30sec
  • (98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles
  • (98C 10sec -> 72C 20sec) X 10-15 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau)
  • 72C 3min
  • Hold 15C

qPCR results

16Hr,4Hr 20Hr,20Hr

  • L:16Hr, 4Hr R:20Hr, 20Hr
  • For 16Hr,4Hr set we should stop at 8 cycles
  • For 20Hr, 20Hr set we should stop the amplification after 7 cycles

PAGE Verification

PAGE

PCR

Components 1x rxn (ul) 12x rxn (ul) Final Conc.
Circularized DNA template 10 0  
KAPA SSYBR FAST MM 2X 50 600 1X
10uM AmpF6.4Sol 2 24 200nM
10uM AmpR6.3IndX 2 0 200nM
H2O 36 432  
Total 100    
  • 98C 30sec
  • (98C 10sec -> 58C 30sec -> 72C 20sec) X 8cycles
  • (98C 10sec -> 72C 20sec) X 10 cycles (should be monitored and stop reaction while it running at 72C before it reaches plateau)
  • 72C 3min
  • Hold 15C
  • Used Index #1-12 for amplification

File:01022014 16hr 4hr.JPG File:01022014 20hr 20hr.JPG

  • L:16Hr, 4Hr R:20Hr, 20Hr

AMPure bead purification protocol

  • Mix 1 volume of bead with PCR product by pipetting 10X
  • Leave at RT for 8min
  • Sit on magnetic 96-ring stand for 5min
  • Wash twice with freshly prepared 200ul 80% EtOH (after adding 80% EtOH, wait for 30sec before pipette up and down few times and discard spnt, and avoid to touch the beads)
  • Dry the beads for 3 min (do not overdry)
  • Resuspend the bead with 50ul EB buffer
  • Leave at RT for 2 min
  • Sit on magnetic 96-ring stand for 3-5min
  • Transfer elute DNA to new tube