Matt:LabNotes/2017-6-19: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
Line 92: Line 92:
| align="right" | 3
| align="right" | 3
| align="right" | 3
| align="right" | 3
| align="right" | 19.4
| align="right" | 0.5
| align="right" | 18.9
| align="right" | 30
| align="right" | 30



Revision as of 22:09, 19 June 2017

Agi15k_Feb2017 V4 + 5% formamide in vitro Capture

Calculate Probes Needed

V4

Probe:target 1000:1 '
Probe size 4998 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 (4998, 157nt) 2.387x10^8 g/mol
Amount Probe req'd 36.7 ng


Probes, Target and Ampligase Buffer Mix

Sample Condition V4 (10.3ng/ul) 12878 DNA (80.3ng/ul) UHRR (1ug/ul) Ampligase Buffer SplintR Buffer Formamide or DMF Ribolock 40U/ul H2O Total
1 Ampligase DNA 3.6 3.8 0 3 0 0 0.5 19.1 30
2 Ampligase NTC 3.6 0 0 3 0 0 0.5 22.9 30
3 SplintR 10% DMF DNA 3.6 3.8 0 0 3 3 0.5 16.1 30
4 SplintR 10% DMF RNA 3.6 0 1 0 3 3 0.5 18.9 30
5 SplintR 10% DMF NTC 3.6 0 0 0 3 3 0.5 19.9 30
6 SplintR 5% formamide DNA 3.6 3.8 0 0 3 1.5 0.5 17.6 30
7 SplintR 5% formamide RNA 3.6 0 1 0 3 1.5 0.5 20.4 30
8 SplintR 5% formamide NTC 3.6 0 0 0 3 1.5 0.5 21.4 30

Program

  • 95C 30sec -> cool down to 55 C at 0.02C/sec -> 55 C 16h