Real-Time Quantitative PCR

Using MasterPLUS SYBR Green I

Kit from ROCHE (# 03 515 869 001)

Prepare your samples (for example ChIP DNA fractions)
Prepare standards for reference curve: 30 ng/μl, 10 ng/μl, 3 ng/μl, 1 ng/μl, 0.3 ng/μl, 0 ng/μl.
Prepare oligo mix at 5 μM each in H20 millipore.

Put 14μl of enzyme (tube 1a) into the mix (tube 1b), PROTECT FROM LIGHT. This ab Mix is stable at -20°C for several days and will enable about 32 reactions.

Prepare Mix for n reactions (PROTECT FROM LIGHT)
H2O from kit:    13 x n μl
Oligo Mix:    2 x n μl
    ab Mix:    4 x n μl

Place capillaries into metal cases, keep track of their numbered position on the cooling block.
Distribute 18.5 μl into each capillary.
Add 1 μl of respective sample to each.
Place CAUTIOUSLY capillaries in microcentrifuge. Spin 15sec at 800 rpm.

Run on LightCycler. Dernière mise à jour : ( 10-12-2007 )