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

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>Noi
 
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* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-6
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-6
== Capture set up (Matt's project) ==
== Capture set up (Matt's project) ==
* 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 ==  
== Probe calculation ==  
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Probe/target'''
| align="center" style="background:#f0f0f0;"|'''Probe:target'''
| align="center" style="background:#f0f0f0;"|'''1000to1'''
| align="center" style="background:#f0f0f0;"|'''1000:1'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
|-
|-
Line 14: Line 25:
| Human gDNA MW||1.9500E+12||g/mol (3E+09 * 650Da/bp+157.9Da)
| Human gDNA MW||1.9500E+12||g/mol (3E+09 * 650Da/bp+157.9Da)
|-
|-
| Human gDNA (300ng)||1.5385E-19||mol
| 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 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)
| 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||1.05E-07||g (6.8151E+08 g/mol * 1.5385E-16mol)
Line 25: Line 36:
|}
|}
* Since I resuspend the probes in too large volume, I concentrate using Qiaquick column, elute in total volume ~62ul, re-quantify conc. ~22ng/ul
* 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
* 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


== 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 mRNA
3-2. ds cDNA no RNasA treatment
4-2. ds cDNA RNaseA treatment
== Probes and AmpLigase Buffer Mix ==
== Probes and AmpLigase Buffer Mix ==
{| {{table}}
{| {{table}}
Line 51: Line 53:
|}
|}


{| {{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;"|'''Targets (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
|-
| 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'''<br>
* 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 ==
{| class="wikitable" style="text-align:center;{{table}} border = 1
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Stock conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Final conc.'''
| align="center" style="background:#f0f0f0;"|'''Unit'''
| align="center" style="background:#f0f0f0;"|'''Prepare volume 30ul'''
|-
|-
| 1-2. gDNA (12878)||4.77||3.00||45.10||6.65||15.58||30.00||299.92
| AmpLigase||5||U/ul||0.5||U/ul||3.00
|-
|-
| 2-2. Total RNA||4.77||3.00||1000.00||1.00||21.23||30.00||1000.00
| 10x AmpLigase Buffer||10||x||1||x||3.00
|-
|-
| 3-2. ds cDNA no RNasA treatment||4.77||3.00||10.20||19.00||3.23||30.00||193.80
| H2O||||||||||24.00
|-
|-
| 4-2. ds cDNA RNaseA treatment||4.77||3.00||7.77||19.00||3.23||30.00||147.63
| Total||||||||||30.00
|}
|}
* Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-3-14

Latest revision as of 21:04, 15 March 2013

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