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

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===GP1_V6===
===GP1_V6===
==Capture setup==
==Capture setup==
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>
|-  align="center"
| width="157" height="13" | GP1_V6
| align="center" width="75" | &nbsp;
| align="center" width="120" | &nbsp;
| align="center" width="75" | &nbsp;
| align="center" width="157" | &nbsp;
| align="center" width="116" | &nbsp;
| align="center" width="75" | &nbsp;
|-  align="center"
| height="13" | Sample
| Conc. (ng/ul)
| Volume for 200ng (ul)
| H2O
| GP1_V6
| 10X ampligase Buffer
| Total
|-  align="center"
| height="13" | CT Jurkat
| align="center" | 47.1
| align="center" | 4.25
| align="center" | 11.47
| align="center" | 2.28
| align="center" | 2
| align="center" | 20
|-  align="center"
| align="center" height="13" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
|-  align="center"
| height="13" | illumina methylation seq pool
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
|-  align="center"
| height="13" | Sample
| Conc. (ng/ul)
| Volume for 200ng (ul)
| H2O
| illumina methylation seq pool
| 10X ampligase Buffer
| Total
|-  align="center"
| height="13" | CT Jurkat
| align="center" | 47.1
| align="center" | 4.25
| align="center" | 10.38
| align="center" | 3.37
| align="center" | 2.00
| align="center" | 20
|-  align="center"
| align="center" height="13" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" |
| align="center" |
| align="center" | &nbsp;
|-  align="center"
| height="13" | ppMALAT1
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
| align="center" | &nbsp;
|-  align="center"
| height="13" | Sample
| Conc. (ng/ul)
| Volume for 200ng (ul)
| H2O
| ppMALAT1
| 10X ampligase Buffer
| Total
|-  align="center"
| height="13" | Jurkat
| align="center" | 100
| align="center" | 2
| align="center" | 15.34
| align="center" | 0.66
| align="center" | 2.00
| align="center" | 20
|-  align="center"
| height="13" | Negative Control
| n/a
| n/a
| align="center" | 17.34
| align="center" | 0.66
| align="center" | 2.00
| align="center" | 20
|}
===illumina methylation seq pool 155 probes setup===
===illumina methylation seq pool 155 probes setup===
===ppMALAT1 setup===
===ppMALAT1 setup===
===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>
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext>

Revision as of 21:32, 16 January 2014

Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set

  • probes prep notes LabNotes/Probe/2014-1-7

[[1]]

Capturing Jurkat gDNA w ppMALT1 ordered form IDT

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

GP1_V6            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O GP1_V6 10X ampligase Buffer Total
CT Jurkat 47.1 4.25 11.47 2.28 2 20
             
illumina methylation seq pool            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O illumina methylation seq pool 10X ampligase Buffer Total
CT Jurkat 47.1 4.25 10.38 3.37 2.00 20
         
ppMALAT1            
Sample Conc. (ng/ul) Volume for 200ng (ul) H2O ppMALAT1 10X ampligase Buffer Total
Jurkat 100 2 15.34 0.66 2.00 20
Negative Control n/a n/a 17.34 0.66 2.00 20

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