Kun:LabNotes/GenomeSeq/2010-8-26

From ZhangLabWiki
Jump to navigation Jump to search

Illumina multiplex library construction[edit]

  • Received sheared DNA from Sara.
  • End-repair:
   Fragmented DNA                              85 μl
   NEBNext End Repair Reaction Buffer (10X)    10 μl
   NEBNext End Repair Enzyme Mix                5 μl
                                               -----
                                              100 μl
   Incubate in a thermal cycler for 30 minutes at 20°C.
   Purified with AMPure beads.
  • A-Tailing:
   Blunt DNA                                42 μl
   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 AMPure beads.
   Conc(ng/ul)  300bp       800bp
   B.Jap-1      22.6        11.7 
   B.Jap-2      14.3        17.6
   B.Jap-3      49.9        58.8
   Ep.G-1       29.5        43.7
   Ep.G-2       25.9   
   Ep.G-3       22.5        74.6
  • Ligation:
                10:1         20:1
   Adaptors     300bp        800bp
   B.Jap-1      1.4ul        0.6ul 
   B.Jap-2      0.9ul        0.8ul
   B.Jap-3      3.2ul        2.8ul
   Ep.G-1       1.9ul        2.1ul
   Ep.G-2       1.6ul   
   Ep.G-3       1.4ul        3.5ul
   End Repaired, Blunt or dA-Tailed DNA     25 μl
   Quick Ligation Reaction Buffer (5X)       8 μl
   20 μM DNA Adaptors                        x μl
   Quick T4 DNA Ligase                     2.5 μl
   H2O                                   4.5-x ul
   Incubate in a thermal cycler for 15 minutes at 20°C. 
   Purified with AMPure beads.    
  • PCR:
  Adapter ligased DNA         10 μl
  PCR_F(10uM)                  2 μl
  PCR_R.N2IndX(10uM)           2 μl  
  50X SYBR Green I           0.8 μl
  H2O                         37 μl
  2X Phusion HF MM            50 μl
                              -----
                             100 μl
   Barcodes     300bp       800bp
   B.Jap-1      Ind1        Ind7 
   B.Jap-2      Ind2        Ind8
   B.Jap-3      Ind3        Ind9
   Ep.G-1       Ind4        Ind10
   Ep.G-2       Ind5   
   Ep.G-3       Ind6        Ind12
  98C 15s ->(98C 5s -> 65C 20s -> 72C 15s)x8 -> (98C 5s -> 72C 20s)x8 -> 72C 2min.
  In each gel, the two lanes on the right are unamplified ligation products.
  File:2010-08-27-library-PCR-2.pngFile:2010-08-27-library-PCR.png
  • Conclusion: library construction didn't work.
  • Possibilities:
    • End-repair/A-tailing reactions didn't work - I still have 30-40ul of sheared DNA
    • Ligation didn't work (adaptor??) - I have ~7ul of ligation-ready DNA
    • PCR didn't work - I have ~25ul of purified ligation products
  • What to do next?
    • Pick two samples (BJ3.3, BJ3.8), repeat the whole protocol with a new kit.
    • Also repeat the ligation of BJ3.3 and BJ3.8 on the remaining ~7ul of DNA with a tube of previously made Y-adaptor.
    • Change the thermal cycling protocol to
 98C 30sec -> (98C 10sec -> 68C 15sec -> 72C 15sec) x 15 cycles.