Noi/NOTES/2013-3-12

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Revision as of 17:11, 12 March 2013 by >Noi (Created page with "== Capture == == Probe calculation == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe/target''' | align="center" style="background:#f0f0f0;"|'''1000to1''' ...")
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Capture

Probe calculation

Probe/target 1000to1 '
Probe size 12,335 probes
Template 300 ng
Human gDNA MW 1.9500E+12 g/mol (3E+09 * 650Da/bp+157.9Da)
Human gDNA (300ng) 1.5385E-19 mol
Probe required (1000:1) 1.5385E-16 mol (1.5385E-19mol x 1000)
Probe MW (12,335 probes, 170nt) 6.8151E+08 g/mol (12335 probes * 170nt * 325Da/bp + 79Da)
Amount probe required 1.05E-07 g (6.8151E+08 g/mol * 1.5385E-16mol)
Amount probe required 105.00 ng
  • Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul

Capture set up

  • Set up capture in 30ul
NTC (no target template)
1-1. gDNA (12878)
2-1. Total mRNA
3-1. ds cDNA no RNasA treatment
4-1. ds cDNA RNaseA treatment
1-2. gDNA (12878)
2-2. Total mRNA
3-2. ds cDNA no RNasA treatment
4-2. ds cDNA RNaseA treatment

Probes and AmpLigase Buffer Mix

Components Volume (ul) 9.5 rxn mix
12K Insitu probes (22ng/ul) 4.77 45.32
10X AmpLigase buffer 3.00 28.50
Total 7.77 73.82
Samples 12K Insitu probes 10X AmpLigase buffer Target template H2O Total
NTC
1-1. gDNA (12878) 4.77 3.00
2-1. Total RNA 4.77 3.00
3-1. ds cDNA no RNasA treatment 4.77 3.00
4-1. ds cDNA RNaseA treatment 4.77 3.00
1-2. gDNA (12878) 4.77 3.00
2-2. Total RNA 4.77 3.00
3-2. ds cDNA no RNasA treatment 4.77 3.00
4-2. ds cDNA RNaseA treatment 4.77 3.00