Noi/NOTES/2013-3-12

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Capture set up (Matt's project)[edit]

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

Probe calculation[edit]

Probe:target 1000:1 '
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 (300ng/1.9500E+12 g/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/nt + 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
  • Note:
    • I showed Matt how to calculate probes and he did calculation, and the number he got agreed with this calculation
    • Even the target amounts in each reaction are not the same, I used the same probe amount in every reaction based on gDNA calculation

Probes and AmpLigase Buffer Mix[edit]

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 Conc. (ng/ul) Targets (ul) H2O (ul) Total (ul) Total amount
in capture (ng)
NTC 4.77 3.00 0 0 22.23 30.00 0.00
1-1. gDNA (12878) 4.77 3.00 45.10 6.65 15.58 30.00 299.92
2-1. Total RNA 4.77 3.00 1000.00 1.00 21.23 30.00 1000.00
3-1. ds cDNA no RNasA treatment 4.77 3.00 15.20 19.00 3.23 30.00 288.80
4-1. ds cDNA RNaseA treatment 4.77 3.00 5.84 19.00 3.23 30.00 110.96
1-2. gDNA (12878) 4.77 3.00 45.10 6.65 15.58 30.00 299.92
2-2. Total RNA 4.77 3.00 1000.00 1.00 21.23 30.00 1000.00
3-2. ds cDNA no RNasA treatment 4.77 3.00 10.20 19.00 3.23 30.00 193.80
4-2. ds cDNA RNaseA treatment 4.77 3.00 7.77 19.00 3.23 30.00 147.63

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 20h
  • -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
  • -> 55 C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.

AmpLigase enzyme mix[edit]

Components Stock conc. Unit Final conc. Unit Prepare volume 30ul
AmpLigase 5 U/ul 0.5 U/ul 3.00
10x AmpLigase Buffer 10 x 1 x 3.00
H2O 24.00
Total 30.00