Kun:LabNotes/CpgSeq/2008-5-14

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cpgMIP28k capture[edit]

  • The primary goal of this experiment is to test whether the probe I made by DpnII/TspRI digestion on the Bst-hRCA amplicon works or not. The probe made on 04/29/08 Exp2 (192nM) with the PCR-USER protocol is used as the positive control.
  • The secondary goal is to compare Stoffel fragment with KlenTaq fragment (ClontTech Titanium Taq).
 SLN mix:
 AmpLigase: 0.5U/ul
 Stoffel or Klentaq: 2U/ul
 dNTP: 10uM
 Reactions                            A            B (KlenTaq)             C                     D
 H2O                                4.5ul        4.5ul                     0ul                 4.5ul
 10X AmpLigase buffer                 1ul          1ul                     1ul                   1ul
 Probe (04/30/08, 192nM)              3ul          3ul   05/06/08(58nM)  7.5ul  03/10/08(218nM)  3ul
 Bis-converted HD50AA (157ng/ul)    1.5ul        1.5ul                   1.5ul                 1.5ul
 SLN mix                              1ul          1ul                     1ul                   1ul
 
 5x (94C2min -> cool down at 0.1C/sec to 55c -> 55C 4h)-> 94C 2min 
   -> 37C 1min -> add 2ul exo I&II -> 37C 2h -> 94C 5min -> 4C hold

PCR[edit]

                                   x4
  Template:                  5ul
  2X iProof master mix:     25ul
  100uM AmpFV6.2           0.2ul
  100uM AmpRV6.2           0.2ul
  50X SYBG I               0.2ul
  H2O                       20ul
  98C 30sec -> 25 x (98C 15sec -> 60C 30sec -> 72C 15sec) -> 72C 3min -> 15C hold.
  File:2008-05-15-CpgMIP-capture-PCR-curves.png
  File:2008-05-15-CpgMIP-capture-PCR-PAGE.png

Conclusion[edit]

  1. Stoffel fragment and KlenTaq are equivalent in terms of activity. KlenTaq is a hot-start enzyme, so it can be added to the reaction mix prior to heat denaturation.
  2. Probes made with the hRCA protocol work fine.