Noi/NOTES/2012-8-26: Difference between revisions
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* Aliquot 24ul, add 1ul of adapter ligated DNA | * Aliquot 24ul, add 1ul of adapter ligated DNA | ||
'''Program''' | '''Program''' | ||
98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x | 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold | ||
[[File:20120826_testqPCR_adapterligated_N37.png| 600px]] | [[File:20120826_testqPCR_adapterligated_N37.png| 600px]] |
Revision as of 04:18, 27 August 2012
WGBS library preparation of low input DNA N37 10 tissues samples using KAPA protocol (from Eppendorf) and epMotion
Preparation of enzyme mix for End repair, A-tailing, Ligation, and Methylation adapters
Sample list
Sample IDs | Sample code on tubes | Well IDs |
N37-Cerebellum (CRBL) | N37-1 | A1 |
N37-Colon | N37-2 | B1 |
N37-Frontal lobe (FL) | N37-3 | C1 |
N37-Heart | N37-4 | D1 |
N37-Small intestine (SI) | N37-5 | E1 |
N37-Liver | N37-6 | F1 |
N37-Lung | N37-7 | G1 |
N37-Skeletal muscle (SM) | N37-8 | H1 |
N37-Pancrease | N37-9 | A2 |
N37-Stomach | N37-10 | B2 |
End repair
Sheared gDNA 200ng in 50ul
Total reaction in | 70 | ul |
DNA | 50 | ul |
End repair reaction mix | 20 | ul |
KAPA End repair mix | 1x rxn | 10x rxn mix |
10X End repai buffer | 7.00 | 70.00 |
KAPA End repair enzyme | 3.00 | 30.00 |
H2O | 10.00 | 100.00 |
Total | 20.00 | 200.00 |
- Aliquot to column #1: 20ul (1 & 2: 40ul)
- Add 20ul of End-repair reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 30min - Add 120ul of diluted AMPure beads - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads
A-tailing
- Total 30ul/reaction
A-tailing reaction mix | 1x rxn | 10x rxn mix |
KAPA 10X A-tailing buffer | 3.00 | 30.00 |
KAPA A-tailing enzyme | 2.00 | 20.00 |
H2O | 25.00 | 250.00 |
Total | 30.00 | 300.00 |
- Aliquot to column #2: 30ul (1 & 2: 60ul)
- Add 30ul of A-tailing reaction mix - Mix by pipetting 20 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 30C for 30min - Add 90ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads
Adapter ligation
Total reaction in | 50 | ul |
Ligation reaction mix | 40 | ul |
Diluted Methylation adapters | 10 | ul |
Ligation reaction mix | 1x rxn | 10x rxn mix |
KAPA 5X Ligation buffer | 10.00 | 100.00 |
KAPA DNA ligase | 3.00 | 30.00 |
H2O | 27.00 | 270.00 |
Total | 40.00 | 400.00 |
- Aliquot ligation reaction mix to column #3: 40ul (1 & 2: 80ul)
Methylation adapter preparation
Prepare adapter | 0.2ulx 10rxn | 2.00 |
H2O | 9.8ulx10rxn | 98.00 |
Total | 100 |
- Aliquot diluted methylation adapter to column #4: 10ul (1 & 2: 20ul)
- Add 40ul of Ligation reaction mix - Mix by pipetting 10 times - Add 10ul of diluted Methylation adapters - Mix by pipetting 10 times - Shake at 1000 rpm for 5min (lid on) - Incubate at 20C for 15min - Add 50ul of 20%PEG/2.5M NaCl - Mix at 750rpm for 5min, 25C (lid on) - Incubate at 25C for 5 min - Put on magnet and discard spnt - Wash twice with 140ul 80% EtOH - Dry the beads - Resuspend adapter ligated DNA with ~23ul EB buffer
qPCR to check if the end repair, A-tailing, and adapter ligation works
Components | 1rxn | 11.5 rxn mix |
Adapter ligated DNA | 1.00 | 0.00 |
10uM PCR_F | 0.50 | 5.75 |
10uM PCR_R.Ind2 | 0.50 | 5.75 |
2x iQ MM | 12.50 | 143.75 |
H2O | 10.50 | 120.75 |
Total | 25.00 | 276.00 |
- Aliquot 24ul, add 1ul of adapter ligated DNA
Program 98C 2min --> (98C 15sec -> 64C 20sec -> 72C 30sec) x 12 cycles --> 72C 3min --> 15C Hold File:20120826 testqPCR adapterligated N37.png