Revision as of 00:39, 22 January 2014 by >Alan6017518
Capturing Bisulfite Converted Jurkat gDNA w GP1 V6 probe set
- probes prep notes LabNotes/Probe/2014-1-7
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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
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Sample
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Conc. (ng/ul)
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Volume for 200ng (ul)
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H2O
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GP1_V6
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10X ampligase Buffer
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Total
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CT Jurkat
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47.1
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4.25
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11.47
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2.28
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2
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20
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illumina methylation seq pool
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Sample
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Conc. (ng/ul)
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Volume for 200ng (ul)
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H2O
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illumina methylation seq pool
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10X ampligase Buffer
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Total
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CT Jurkat
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47.1
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4.25
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10.38
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3.37
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2.00
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20
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ppMALAT1
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Sample
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Conc. (ng/ul)
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Volume for 200ng (ul)
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H2O
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ppMALAT1
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10X ampligase Buffer
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Total
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Jurkat
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100
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2
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15.34
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0.66
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2.00
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20
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Negative Control
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n/a
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n/a
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17.34
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0.66
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2.00
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20
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illumina methylation seq pool 155 probes setup
Program stored under Alan/aCPG
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 58C 20h
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
GP1_V6 & ppMALAT1 setup
Program stored under Kun/CPG
-> 95c 30sec -> cool down to 55C at 0.02C/sec -> 55C 20h
-> add 3ul SLN mix (2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 100uM dNTP)
-> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 2min -> 4C hold.
Prepare SLN Mix
SLN Mix
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Components
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Stock Concentration
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Unit
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Final Concentration
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Unit
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Prepare Volume (ul)
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AmpliTaq Stoffel
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10
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U/ul
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2
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U/ul
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2.6
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Ampligase
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5
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U/ul
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0.5
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U/ul
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1.3
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dNTP
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1
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mM
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100
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uM
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1.3
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10X Ampligase Buffer
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10
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X
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1
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X
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1.3
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H2O
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n/a
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n/a
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n/a
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n/a
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6.5
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Total
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n/a
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n/a
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n/a
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n/a
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13
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Prepare Exo Mix
- Mix 1:1 Exo I & Exo III (9ul total)
PCR Amplification
(AmpF6.4Sol and AmpR6.3 for GP1_V6 & illumina_155)
(ISB_CA_AF and ISB_CA_AR_T2 for ppMALTA1)
Probes
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BSPP_GP1_V6
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illumina_155
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Sample
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Jurkat_CT
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Jurkat_CT
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Index
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1
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2
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Components
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1x rxn
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1x rxn
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Captured Template
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20
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20
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10uM AmpF6.4Sol
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1
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1
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10uM AmpR6.3Indx
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1
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1
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2X KAPA SYBR MM
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25
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25
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H2O
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3
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3
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Probes
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ppMALTA1
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ppMALTA1
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Sample
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Jurkat_gDNA
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-ve control
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Components
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1x rxn
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1x rxn
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Captured Template
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20
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20
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10uM ISB_CA_AF
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1
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1
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10uM ISB_CA_AR_T2
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1
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1
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2X KAPA SYBR MM
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25
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25
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H2O
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3
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3
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PAGE Gel