Noi/NOTES/2013-3-12: Difference between revisions

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{| {{table}} border = 1
{| class="wikitable" style="text-align:center;{{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''12K Insitu probes'''
| align="center" style="background:#f0f0f0;"|'''12K Insitu probes'''
| align="center" style="background:#f0f0f0;"|'''10X AmpLigase buffer'''
| align="center" style="background:#f0f0f0;"|'''10X AmpLigase buffer'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Conc. (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Target template'''
| align="center" style="background:#f0f0f0;"|'''Target template<br>(ul)'''
| align="center" style="background:#f0f0f0;"|'''H2O'''
| align="center" style="background:#f0f0f0;"|'''H2O (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total (ul)'''
| align="center" style="background:#f0f0f0;"|'''Total amount in capture (ng)'''
| align="center" style="background:#f0f0f0;"|'''Total amount <br>in capture (ng)'''
|-
|-  
| NTC||4.77||3.00||0||0||22.23||30||0.00
| 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
| 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
| 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
| 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
| 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
| 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
| 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
| 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
| 4-2. ds cDNA RNaseA treatment||4.77||3.00||7.77||19.00||3.23||30.00||147.63
|}
|}

Revision as of 18:41, 12 March 2013

Capture set up (Matt's project)

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
  • Note: Even the target amounts in each reaction are not the same, I used the same probe amount in every reaction based on gDNA calculation

Capture set up

  • Set up capture in 30ul
NTC (no target template)
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

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 Conc. (ng/ul) Target template
(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