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

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===Probes, Target and Ampligase Buffer Mix===
===Probes, Target and Ampligase Buffer Mix===
*gDNA: 12878 (80ng/ul)
*gDNA: 12878 (80.3ng/ul)
*[[Matt:LabNotes/2015-1-12 | First strand cDNA from UHRR]]
*[[Matt:LabNotes/2015-1-12 | First strand cDNA from UHRR]]
*[[Matt:LabNotes/2014-11-19#Final_Probes_to_Order:_Trim_to_12.2C000_probes | CA12k_Nov2014 Probes]]
*[[Matt:LabNotes/2014-11-19#Final_Probes_to_Order:_Trim_to_12.2C000_probes | CA12k_Nov2014 Probes]]
**[[Matt:LabNotes/2015-1-5#TBU_Gel_Quantification | V4 (10.7 ng/ul)]]
**[[Matt:LabNotes/2015-1-5#TBU_Gel_Quantification | V4 (10.7 ng/ul)]]
**[[Matt:LabNotes/2015-1-15#TBU_Gel_Quantification | V7 (39.2 ng/ul)]]
**[[Matt:LabNotes/2015-1-15#TBU_Gel_Quantification | V7 (39.2 ng/ul)]]
***Dilute 1ul into 2ul for easier more accurate pipetting
***Dilute 2ul into 4ul for easier more accurate pipetting


{| {{table}}
{| {{table}}
Line 65: Line 65:
| 2||V4 - cDNA||2.5||3.8||3||20.7||30
| 2||V4 - cDNA||2.5||3.8||3||20.7||30
|-
|-
| 3||V7 - gDNA||1||15||3||11||30
| 3||V4 - NTC||2.5||0||3||24.5||30
|-
|-
| 4||V7 - cDNA||1||15||3||11||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
|}
|}



Revision as of 01:57, 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.