Kun:LabNotes/GenomeSeq/2010-9-9

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Repeat the Illumina library construction on the remaining DNA[edit]

  • In this experiment, I used the adaptors that Alice prepared. I also reduce the AMPure beads to 1 volume of the reaction (eliminated small DNA fragments).
  • End-repair:
   Fragmented DNA                              30 μl
   NEBNext End Repair Reaction Buffer (10X)   3.5 μl
   NEBNext End Repair Enzyme Mix              1.5 μl
                                               -----
                                              35 μl
   Incubate in a thermal cycler for 30 minutes at 20°C.
   Purified with Qiaquick columns, eluted with 30ul EB
  
   Concentration (ng/ul) 
   BJ1.3   28          BJ1.8    16
   BJ2.3   20          BJ2.8    19
   EP1.3   27          EP1.8    35
   EP2.3   26         
   EP3.3   27          EP3.8    38
   1A      16          1G       13
   1F      12
  • A-Tailing:
   Blunt DNA                                   28 μl
   NEBNext dA-Tailing Reaction Buffer (10X)   3.5 μl
   Klenow Fragment (3´→ 5´ exo–)              2.5 μl
   H2O                                          1 μl
                                               -----
                                               35 μl
   Incubate in a thermal cycler for 30 minutes at 37°C.
   Purified with MinElute columns, eluted with 20ul H2O.
   
   Concentration (ng/ul) 
   BJ1.3   20          BJ1.8    12
   BJ2.3   17          BJ2.8    16
   EP1.3   28          EP1.8    35
   EP2.3   30         
   EP3.3   24          EP3.8    51
   1A      11          1G       6
   1F       6    

  • Ligation:
   Adaptors (20uM) adaptor:insert=1:20
   BJ1.3   2.0          BJ1.8    0.5
   BJ2.3   1.7          BJ2.8    0.6
   EP1.3   2.8          EP1.8    1.3
   EP2.3   3.0         
   EP3.3   2.4          EP3.8    2.0
   1A      1.1          1G       0.6
   1F      0.6     
   End Repaired, Blunt or dA-Tailed DNA     18 μl
   Quick Ligation Reaction Buffer (5X)       6 μl
   20 μM DNA Adaptors                        x μl
   Quick T4 DNA Ligase                       2 μl
   H2O                                     4-x ul
   Incubate RT for 15 minutes. 
   Purified with 1V AMPure beads.    
  • PCR:
  Adapter ligased DNA         10 μl
  PCR_Fx(100uM)              0.2 μl
  PCR_R(10uM)                  2 μl  
  50X SYBR Green I           0.8 μl
  H2O                         37 μl
  2X Phusion HF MM            50 μl
                              -----
                             100 μl
   
 98C 30sec -> (98C 10sec -> 65C 20sec -> 72C 20sec) x 8 cycles. 
              -> (98C 10sec -> 72C 20sec) x 4 cycles.
 File:2010-09-09-ligation-PCR1.png  File:2010-09-09-ligation-PCR2.png
  • Conclusion: All these libraries worked, indicating that somehow the adaptors I used in the previous experiments are problematic.