Rui:LabNotes/Haplotyping/2014-12-2

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Revision as of 08:32, 4 December 2014 by >RuiLiu (→‎Procedure)
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Purpose

  • repeat selector procedure:
  • selector : gDNA = 200:1; linker : selector = 3:1
  • direct gDNA input: 12, 24, 48ng
  • compare selector vs padlock probe
  • premade

Samples

  • HapMap gDNA from Coriell GM18506 (diluted to 50ng/ul)
  gDNA input Selector set Probe ratio Indx Test
#1 48ng linkerV2_UMI 200:1; 3:1 Indx01 selector_UMI
#2 24ng linkerV2_UMI 200:1; 3:1 Indx02 selector_UMI
#3 12ng linkerV2_UMI 200:1; 3:1 Indx03 selector_UMI
#4 NTC linkerV2_UMI 200:1; 3:1 Indx04 selector_UMI
#5 48ng linkerV2 200:1; 3:1 Indx05 selector
#6 24ng linkerV2 200:1; 3:1 Indx06 selector
#7 12ng linkerV2 200:1; 3:1 Indx07 selector
#8 NTC linkerV2 200:1; 3:1 Indx08 selector
#9 48ng linkerV2 200:1 Indx09 padlock probe
#10 24ng linkerV2 200:1 Indx10 padlock probe
#11 12ng linkerV2 200:1 Indx11 padlock probe
#12 NTC linkerV2 200:1 Indx12 padlock probe
#13 50ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx13 selector_UMI
#14 20ng_digested (Kun's, asPC) linkerV2_UMI 200:1; 3:1 Indx14 selector_UMI

Procedure

1a. R.E digestion

Components 48ng 24ng 12ng NTC
gDNA (12ng/ul) 4.00 2.00 1.00 0.00
10x cutsmart buffer 1.00 1.00 1.00 1.00
DpnII (50U/ul) -> dilute to 5U/ul 2.50 1.25 0.63 0.63
BfaI (10U/ul) -> dilute to 2U/ul 2.50 1.25 0.63 0.63
H2O 0.00 4.50 6.75 7.75
Total 10.00 10.00 10.00 10.00
Temperature Duration
Digestion 37C, 1hr
hold 4C
Components Volume (ul) Final conc.
10X NEBuffer 3.1 1 1x
Temperature Duration
Digestion 37C, 30min
heat inactivation 80C, 20min
hold 4C

1b. Assembly of selecotr and linker