Kun:LabNotes/Haplotyping/2010-8-26: Difference between revisions

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m (Created page with '==Illumina multiplex library construction== *Received sheared DNA from Sara. *End-repair: Fragmented DNA 85 μl NEBNext End Repair Reaction B…')
 
 
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     Incubate in a thermal cycler for 30 minutes at 37°C.
     Incubate in a thermal cycler for 30 minutes at 37°C.
     Purified with AMPure beads.
     Purified with AMPure beads.
              Conc(ng/ul)
    1A          17.7
    1F          11.8
    1G          7.3
    2E          3.1
    2F          5.1
*Ligation:
*Ligation:
     End Repaired, Blunt or dA-Tailed DNA    27 μl
     End Repaired, Blunt or dA-Tailed DNA    25 μl
     Quick Ligation Reaction Buffer (5X)      8 μl
     Quick Ligation Reaction Buffer (5X)      8 μl
     20 μM DNA Adaptors                     2.5 μl
     20 μM DNA Adaptors                       2 μl
     Quick T4 DNA Ligase                    2.5 μl
     Quick T4 DNA Ligase                    2.5 μl
    H2O                                    2.5 μl
     Incubate in a thermal cycler for 15 minutes at 20°C.  
     Incubate in a thermal cycler for 15 minutes at 20°C.  
     Purified with AMPure beads.     
     Purified with AMPure beads.     
Line 34: Line 41:
                               100 μl
                               100 μl
     Barcodes    200bp
     Barcodes    200bp
     1A          Ind8
     1A          Ind10
     1F          Ind9
     1F          Ind11
     1G           Ind10
     1G          Ind12
    2E          Ind11
    2F           Ind12
   98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min.
   98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min.
   Terminated the reactions before the curves reach the plateau stage.
   Terminated the reactions before the curves reach the plateau stage.
*The libraries were not good. See [[Kun:LabNotes/GenomeSeq/2010-8-26]]
*Repeated the library construction, and was able to make three good libraries: [[Kun:LabNotes/GenomeSeq/2010-9-10]]
*Obtained two lanes of SE 60bp reads from HL072 run.
        Data file              # reads
  GM20431_chr_1A_sequence.txt 7,613,296
  GM20431_chr_1F_sequence.txt 7,760,883
  GM20431_chr_1G_sequence.txt 6,546,229

Latest revision as of 00:57, 18 September 2010

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) 
   1A           17.7
   1F           11.8
   1G           7.3
   2E           3.1
   2F           5.1
  • Ligation:
   End Repaired, Blunt or dA-Tailed DNA     25 μl
   Quick Ligation Reaction Buffer (5X)       8 μl
   20 μM DNA Adaptors                        2 μl
   Quick T4 DNA Ligase                     2.5 μl
   H2O                                     2.5 μl
   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     200bp
   1A           Ind10
   1F           Ind11
   1G           Ind12
  98C 15s ->(98C,5s -> 65C,20s -> 72C,15s)x15 -> 72C 2min.
  Terminated the reactions before the curves reach the plateau stage.
       Data file               # reads
  GM20431_chr_1A_sequence.txt	7,613,296
  GM20431_chr_1F_sequence.txt	7,760,883
  GM20431_chr_1G_sequence.txt	6,546,229