AlanFung:LabNotes/Capturing/2014-1-2

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Helping Dheeraj to test Klentaq capture reactions[edit]

  • 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[edit]

PCR amplification to check if capture works[edit]

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[edit]

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[edit]

PAGE

PCR[edit]

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[edit]

  • 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

PAGE Gel Size Selection[edit]