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

From ZhangLabWiki
Jump to navigation Jump to search
Line 26: Line 26:
     Ep.G-3      Ind6        Ind6+12
     Ep.G-3      Ind6        Ind6+12
     1A          Ind1+7
     1A          Ind1+7
     1F          Ind2+9
     1F          Ind2+10(?)
     1G          Ind3+11
     1G          Ind3+11
   98C 30 ->(98C 10s -> 65C 20s -> 72C 20s)x8 -> (98C 10s -> 72C 20s)x4 -> 72C 2min.
   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.
   Took all except BJ2.3/BJ3.3/BJ2.8/BJ3.8 out after 9 cycles.
  [[Image:2010-09-10-ligation-PCR.png|350px]]
 
   Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O
   Purified 50ul of each amplicon with 50ul AmPure beads, eluted with 40ul H2O
     Conc(ng/ul)  300bp      800bp
     Conc(ng/ul)  300bp      800bp
Line 42: Line 43:
     1F          5
     1F          5
     1G          6
     1G          6
   
    Combine
    B.Jap1.3, B.Jap2.3, B.Jap3.3, EP1.3, EP2.3, EP3.3 => Lancelet 300bp library
    B.Jap1.8, B.Jap2.8, B.Jap3.8, EP1.8, EP3.8 => Lancelet 800bp library
    1A, 1F, 1G => GM20431 Chr. library
    Perform size selection, cut out two different bands from the Lancelet 800bp library.
    Estimate size:
        Lancelet-300bp: 250-350bp
        Lancelet-800bp: 900-1000bp
        Lancelet-600bp: 600-700bp
    [[Image:2010-09-10-Lancelet-300bp-lib-size-selection.png|400px]]
    [[Image:2010-09-10-Lancelet-800bp-lib-size-selection.png|400px]]
    [[Image:2010-09-10-GM20431-chr-lib-size-selection.png|400px]]

Revision as of 00:56, 11 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+10(?)
   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.
  File:2010-09-10-ligation-PCR.png
  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
   
   Combine 
   B.Jap1.3, B.Jap2.3, B.Jap3.3, EP1.3, EP2.3, EP3.3 => Lancelet 300bp library
   B.Jap1.8, B.Jap2.8, B.Jap3.8, EP1.8, EP3.8 => Lancelet 800bp library
   1A, 1F, 1G => GM20431 Chr. library
   Perform size selection, cut out two different bands from the Lancelet 800bp library. 
   Estimate size: 
       Lancelet-300bp: 250-350bp
       Lancelet-800bp: 900-1000bp
       Lancelet-600bp: 600-700bp
   File:2010-09-10-Lancelet-300bp-lib-size-selection.png
   File:2010-09-10-Lancelet-800bp-lib-size-selection.png
   File:2010-09-10-GM20431-chr-lib-size-selection.png