Matt:LabNotes/2015-1-21: Difference between revisions

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* -> add 3ul AmpLigase enzyme mix (0.5U/ul AmpLigase in 1X AmpLigase buffer)
* -> 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.
* -> 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'''
|-
| AmpLigase||5||U/ul||0.5||U/ul||2.00
|-
| 10x AmpLigase Buffer||10||x||1||x||2.00
|-
| H2O||||||||||16.00
|-
| Total||||||||||20.00
|}

Revision as of 21:59, 22 January 2015

CA12k_Nov2014 V4 and V7 in vitro Capture

Calculate Probes Needed

V4

Probe:target 1000:1 '
Probe size 3514 probes
DNA templet 300 ng
gDNA MW 1.95x10^12 g/mol
gDNA (300ng) 1.5385x10^-19 mol
Probe (1000:1) 1.5385x10^-16 mol
Probe MW (3514, 150nt) 1.71585106x10^8 g/mol
Amount Probe req'd 26.4 ng

V7

Probe:target 1000:1 '
Probe size 2486 probes
DNA templet 300 ng
gDNA MW 1.95x10^12 g/mol
gDNA (300ng) 1.5385x10^-19 mol
Probe (1000:1) 1.5385x10^-16 mol
Probe MW (2486, 150nt) 1.21388894x10^8 g/mol
Amount Probe req'd 18.7 ng

Probes, Target and Ampligase Buffer Mix

Sample # Sample Description Probes Target 10X Ampligase Buffer H2O Total
1 V4 - gDNA 2.5 3.8 3 20.7 30
2 V4 - cDNA 2.5 3.8 3 20.7 30
3 V4 - NTC 2.5 0 3 24.5 30
4 V7 - gDNA 1 15 3 11 30
5 V7 - cDNA 1 15 3 11 30
6 V7 - NTC 1 0 3 26 30

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

Components Stock conc. Unit Final conc. Unit Prepare volume 30ul
AmpLigase 5 U/ul 0.5 U/ul 2.00
10x AmpLigase Buffer 10 x 1 x 2.00
H2O 16.00
Total 20.00