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.

File:10-20-10 gel.jpeg