Kun:LabNotes/GenomeSeq/2010-9-10: Difference between revisions

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>Sam Chiang
Line 10: Line 10:
     Purified with 1V AMPure beads.     
     Purified with 1V AMPure beads.     
===PCR===
===PCR===
   Adapter ligated DNA        10 μl
   Adapter ligated DNA        20 μl
   PCR_F(10uM)                  2 μl
   PCR_F(10uM)                  2 μl
   PCR_R.N2IndX(10uM)          2 μl   
   PCR_R.N2IndX(10uM)          2 μl   
   50X SYBR Green I          0.8 μl
   50X SYBR Green I          0.8 μl
   H2O                        37 μl
   H2O                        25 μl
   2X Phusion HF MM            50 μl
   2X Phusion HF MM            50 μl
                               -----
                               -----

Revision as of 21:48, 10 September 2010

Library construction (cont.)

  • The libraries made on 9/9 were good. I need to repeat the ligation using the ligation products made on 8/26 for BJ3.3 and BJ3.8. Then I will do ligation PCR with the barcoded primers on all the ligation products.

Ligation

   Purified ligation products from 8/36     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 ligated DNA         20 μl
  PCR_F(10uM)                  2 μl
  PCR_R.N2IndX(10uM)           2 μl  
  50X SYBR Green I           0.8 μl
  H2O                         25 μl
  2X Phusion HF MM            50 μl
                              -----
                             100 μl
   Barcodes     300bp       800bp
   B.Jap-1      Ind1        Ind1+7 
   B.Jap-2      Ind2        Ind2+8
   B.Jap-3      Ind3        Ind3+9
   Ep.G-1       Ind4        Ind4+10
   Ep.G-2       Ind5   
   Ep.G-3       Ind6        Ind6+12
   1A           Ind1+7
   1F           Ind2+9
   1G           Ind3+11
  98C 30 ->(98C 10s -> 65C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x4 -> 72C 2min.
  Took all except BJ2.3/BJ3.3/BJ2.8/BJ3.8 out after 9 cycles.

  Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O
   Conc(ng/ul)  300bp       800bp
   B.Jap-1      12           6 
   B.Jap-2      12           9
   B.Jap-3      3            5
   Ep.G-1       10           8
   Ep.G-2       4   
   Ep.G-3       7            7
   1A           8
   1F           5
   1G           6