AlanFung:LabNotes/Capturing/2014-1-14: Difference between revisions

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>Alan6017518
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===GP1_V6 setup===
===GP1_V6 setup===
==Prepare SLN Mix==
==Prepare SLN Mix==
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|-  align="center"
| width="117" height="13" | SLN Mix
| align="center" width="110" | &nbsp;
| align="center" width="75" | &nbsp;
| align="center" width="75" | &nbsp;
| align="center" width="75" | &nbsp;
| align="center" width="109" | &nbsp;
|-  align="center"
| align="center" height="13" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
|-  align="center"
| height="13" | Components
| Stock Concentration
| Unit
| Final Concentration
| Unit
| Prepare Volume (ul)
|-  align="center"
| height="13" | AmpliTaq Stoffel
| align="center" | 10
| U/ul
| align="center" | 2
| U/ul
| align="center" | 2.6
|-  align="center"
| height="13" | Ampligase
| align="center" | 5
| U/ul
| align="center" | 0.5
| U/ul
| align="center" | 1.3
|-  align="center"
| height="13" | dNTP
| align="center" | 1
| mM
| align="center" | 100
| uM
| align="center" | 1.3
|-  align="center"
| height="13" | 10X Ampligase Buffer
| align="center" | 10
| X
| align="center" | 1
| X
| align="center" | 1.3
|-  align="center"
| height="13" | H2O
| n/a
| n/a
| n/a
| n/a
| align="center" | 6.5
|-  align="center"
| height="13" | Total
| n/a
| n/a
| n/a
| n/a
| align="center" | 13
|}

Revision as of 19:09, 16 January 2014

Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set

Capturing Jurkat gDNA w ppMALT1

Bisulfite Conversion of Jurkat gDNA

  • Bisulfite conversion with zymo methylation gold
  • Convert 500ng of Jurkat gDNA (2 rxn, 100ng total)
  • Add 130ul of lighting conversion reagent to 5ul of jurkat gDNA and 15ul of ddH2O
  • Mix and centrifuge
1. 98°C for 8 minutes 
2. 54°C for 60 minutes 
3. 4°C hold
  • Add 600ul M-binding buffer to column
  • load the sample into column, mix by inverting column and centrifuge at 14000rpm for 30sec
  • discard flow through
  • add 100ul m-wash buffer to the column and centrifuge at 14000rpm for 30sec
  • add 200ul L-Desulphonation buffer to the column and sit at RT for 20 minutes and centrifuge at 14000rpm for 30sec
  • add 200ul of m-wash buffer to the column and centrifuge at 14000rpm for 30sec
  • Add 10ul of EBuffer to column sit for 5 mintues and centrifuge at 14000rpm for 30sec (2 rxn total)

ssDNA Qubit assay

  • 47.1ng/ul *20ul = 942ng
  • Stored in Maytag -20C (Alan Misc. #9)

Probe calculations

illumina methylation seq pool 155

ppMALAT1

GP1_V6

Capture setup

illumina methylation seq pool 155 probes setup

ppMALAT1 setup

GP1_V6 setup

Prepare SLN Mix

SLN Mix          
           
Components Stock Concentration Unit Final Concentration Unit Prepare Volume (ul)
AmpliTaq Stoffel 10 U/ul 2 U/ul 2.6
Ampligase 5 U/ul 0.5 U/ul 1.3
dNTP 1 mM 100 uM 1.3
10X Ampligase Buffer 10 X 1 X 1.3
H2O n/a n/a n/a n/a 6.5
Total n/a n/a n/a n/a 13