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]
- 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.
- Probes made with the hRCA protocol work fine.