Jeff:LabNotes/Microbiome/2010-10-20
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Haplotyping Library construction[edit]
Use sheared DNA from 10-19-10
- Only have a 50 uL volume
- End-repair:
Fragmented DNA 50 μl NEBNext End Repair Reaction Buffer (10X) 6 μl NEBNext End Repair Enzyme Mix 3 μl Water 1 ul ----- 60 μl Incubate in a thermal cycler for 30 minutes at 20°C. Purified with qiaquick with 40 uL elution volume.
Conc(ng/ul) 1B 21.4 1C 13.9 1D 45.1 1G 17.1
- A-Tailing:
Blunt DNA 40 μl Water 2 ul NEBNext dA-Tailing Reaction Buffer (10X) 5 μl Klenow Fragment (3´→ 5´ exo–) 3 μl ----- 50 μl Incubate in a thermal cycler for 30 minutes at 37°C. Purified with quiquick (40 uL elution volume). Conc(ng/ul) 1B 18.6 1C 20.9 1D 39.9 1G 14.1
- Ligation:
End Repaired, Blunt or dA-Tailed DNA 20 μl Quick Ligation Reaction Buffer (5X) 6 μl 20 μM DNA Adaptors x μl Quick T4 DNA Ligase 1 μl H2O 3-x μl Incubate in a thermal cycler for 15 minutes at 20°C. Purified with AMPure beads.
Use the following to calculate x ul adaptors
y=(__ M DNA *40 uL volume)/(300 bp*660 Da)
x=y*15/20 uM
Conc(ng/ul) 1B 30.9 ? 1C 9.6 1D 14.9 1G 5.6
Haplotype: PCR of LIbraries
Run PCR on libraries made today.
Create the following mix
- PCR: 1 rxn Master (13 rxn)
Adapter ligased DNA 5 μl --- PCR_F(10uM) 2 μl 26 PCR_R.N2IndX(10uM) 2 μl --- 50X SYBR Green I 0.8 μl 10.4 H2O 40 μl 520 2X Phusion HF MM 50 μl 650 ---- 100
- Add 93 uL of master mix to 12 tubes
- Add 5 ul ligated DNA (B, C, D, G) to 3 tubes each
- For B, add 2 uL of reverse primer 1, 2, and 3 to the separate tubes. C use 4,5,6. D use 7,8,9. G use 10,11,12.
- Run the following program: 98 C for 30 s -> (98C 10s, 65C 20s, 72C 20s)x8 -> (98C 10s, 72C 20s)x5 -> 72C 2 min.
Results
I stopped after 8 cycles and it still seems to be overamplified. I will have to repeat and stop earlier.