Kun:LabNotes/ExonomeSeq/2008-1-8

From ZhangLabWiki
Jump to navigation Jump to search

Exp.1 Perform 2nd round PCR on the PAGE purified 450bp band from the previous capturing experiment.[edit]

                         dNTP       dUTP
    Template:            5ul       5ul
    10X PCR buffer:     10ul      10ul
    10mM dNTP:           2ul       2ul
    1mM dUTP:            0ul       1ul
    100uM AmpFSolV6.2  0.4ul     0.4ul
    100uM AmpRSolV6.2  0.4ul     0.4ul
    50X SYBG I:        0.4ul     0.4ul
    JumpStart Taq:       2ul       2ul
    H2O:                80ul      79ul

94C 3min -> (94C 30sec -> 72C 1min) x 10cyc -> 72C 3min -> 15C hold. Purify 50ul with Bioneer columns. dNTP: 31ng/ul x 30ul dUTP: 30ng/ul x 30ul

Exp.2 Make sequencing library by USER digestion and ligation[edit]

15ul dUTP amplicon -> add 1.5ul USER enzyme -> 37C 1h -> set up ligation.

Exp. 3 Make sequencing library by random priming[edit]

                       SolF/Rn6     N6
     DNA                  5ul       5ul
     10X Klenow buffer  2.5ul     2.5ul
     100uM SolFn6      0.25ul       0ul
     100uM SolRn6      0.25ul       0ul
     1mM N6               0ul     0.5ul
     10mM dNTP            1ul       1ul
     H2O                 14ul      14ul

94C 2min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> 94C 1min -> 16C 1min -> add 1ul 20U/ul Klenow exo- -> 16C 10min -> 37C 10min -> purified with MinElute columns.

Setup ligation for the N6 reaction.

Exp.4 Perform another round of exon capture experiment[edit]

   Sheared NA10835(~300ng/ul)                3ul
   E55kv1 probes 107nM (01/07/08,purified)  17ul
   10X Ampligase buffer                   2.25ul

95C 10min -> 60C 40h -> add 2.5ul NSL mix (Stoffel 1U/ul, AmpLigase 0.5U/ul, dNTP 5uM) -> 60C 1h -> (95C 2min -> 60C 1h) x 20 -> 37C 1min

Not sure I added the right NSL mix or the one for SNP . So I re-started the circularization reaction: 95C 5min -> 60C 20h -> add 2.5ul NSL mix -> 60C 1h -> 37C 1min -> add 2ul exo I&III, 1ul T7 exo -> 37C 2h -> 95C 2min -> 4c hold

Exp.5 Another round of probe prep for E55kv1 and CES22k #5[edit]

PCR (with NEB Taq master mix)

                             x1          x25     
        E55k1(20nM)         0.1ul       2.5ul     CES22k #5(100nM)  0.5
        2x Taq master mix    50ul      1250ul                     1250ul 
        100uM pAP1V6U       0.4ul        10ul                       10ul 
        100uM phosAP2V6     0.4ul        10ul                       10ul 
        H2O                49.3ul      1230ul                     1230ul

94C 3min -> 20 cycles of (94C 45sec -> 58C 2min -> 72C 1min) -> 72C 3min -> 15C hold

Ethanol precipitation.

Resuspended in 170ul dH2O, add 20ul 10X Lambda exo buffer, 10ul 5U/ul lambda exo, 37C 3h -> 90C 10min.

Purify 50ul with MinElute columns. Measure concentration:

  • CES22k #5: 98ng/ul x 20ul
  • E55kv1: 71ng/ul x 20ul

Added 2ul USER enzyme, 37C overnight. Added 20ul dH2O, 5ul 10X Dpn II buffer, 2ul RE-DpnIIs guide oligo. 95C 10min -> turn off the heater, let the reaction slowly cool down in ~15 minutes -> add 4ul Dpn II -> 37C 1 hour -> add 4ul USER enzyme -> 37C 2 hour.

Clean up the remaining 150ul ssDNA with G25 column, add 10ul USER enzyme, 37C 2h -> add 20ul Dpn II buffer, 6ul RE-Dpn IIs guide oligo. 95C 10min -> turn off the heater, let the reaction slowly cool down in ~15 minutes -> add 12ul Dpn II -> 37C 1 hour -> add 8ul USER enzyme -> 37C 2 hour.

50ul of the first batch digestion + 50ul of the second batch digestion -> PAGE purification with one 2D TBU gel.

Phenol/chloroform extraction of the remaining 150ul second batch digestion. -> PAGE purification of 50ul with 2D TBU gel.

Yield:

  • E55kv1, no phenol extraction: 5.1ng/ul => 180nM x 30ul
  • E55kv1, phenol extraction: 7.8ng/ul => 275nM x 30ul
  • CES22k #5, no phenol extraction: 6.4ng/ul => 246nM x 30ul
  • CES22k #5, phenol extraction: 9.3ng/ul => 358nM x 30ul