Kun:LabNotes/CpgSeq/2007-12-11

From ZhangLabWiki
Jump to navigation Jump to search

Exp.1 Repeat the PCR experiment on the circularized reactions using Hd48MIP1,2,5 primers with a touchdown protocol[edit]

   Template:            1ul  (1:100 dilution of bifulfite converted gDNAs)
   10X buffer:        2.5ul
   10mM dNTP:         0.5ul
   10uM Primer pair  0.75ul
   50X SYBR I         0.2ul
   AmpliTaq Gold     0.25ul
   H2O                 20ul

Total number of reactions: 4 MIP reactions x 3 primer pairs + 2 control x 3 primer pairs = 18 95C 15min -> 8 cycles of (94C 30sec -> 60C 30sec (-0.5C per cycle) -> 72C 30 sec) -> 27 cycles of (94C 30sec -> 56C 30sec -> 72C 30 sec) -> 72C 3min -> 15C hold.

  File:2007-12-11-CpG-MIP-PCR-specific-primer-touchdown.png

Exp.2 Perform another round of capturing reaction at 60C[edit]

  1. dsDNA synthesis:
    DNA (11/12/07)      20ul
    10x NEBbuffer2       3ul
    10mM dNTP          1.5ul
    100uM N6             3ul
    

94C 3min -> snap on ice for >1min -> add 2ul 10U/ul DNA Pol I -> 16C 15min -> 37C 30min -> 75C 10min. Purify with MinElute columns, eluted with 22ul ddH2O. Measure concentration.

               RL dsDNA(47ng/ul) Jurkat dsDNA(95ng/ul)
    Template      10ul                  5ul
    10x buffer   1.5ul                1.5ul
    10nM CpG-MIP   2ul                  2ul       (set up another 2 with the v1 probes)
    H2O          1.5ul                6.5ul
              RL Bis-DNA            Jurkat Bis-DNA
    Template       5ul                  5ul
    10x buffer   1.5ul                1.5ul
    10nM CpG-MIPv2 2ul                  2ul      
    H2O          6.5ul                6.5ul


              Jurkat gDNA           Jurkat gDNA (100ng/ul)
    Template       5ul                  5ul
    10x buffer   1.5ul                1.5ul
    10nM CpG-MIPv1 2ul     CpG-MIPv1    2ul      
    H2O          6.5ul                6.5ul

95C 3min->60C 42h-> add 1ul SLN mix (2U/ul stoffel, 2U/ul AmpLigase, 1mM dNTP) -> 65C 1h -> 40 cycles of (95C 1min -> 65C 15min) -> 65C 4h -> 37C 1min -> add 1ul of exo mix (Exo I, Exo III, T7 exo), 0.5ul USER enzyme->37c 1 hour -> 94C 5min -> 4C hold.