Analysis of nucleic acids

Quantitative PCR 1- PCR efficiency

Do a serial dilution of RT product or DNA sample (usually 1/10 step is fine – lower steps can be use, especially for RT if the target RNA is not highly expressed. Alternatively, approximately 107 copies of a plasmid containing the target sequence can be added to the RT product)

2- Light Cycler kit

Dilute your sample at appropriated dilution. This one has been determine during the setup experiment.

Protocol: PCR Mix‡‡‡

1µl

25mM MgCl2

X µL§§§

10µM forward primer

0.5-1µL****

10µM reverse primer

0.5-1µL

PCR-grade H20

Y µL (+ 0.5% of final volume)

This mix can be prepared before and kept at 4°C

Mix 5µl of PCR master mix with 5µL of diluted RT product (not enough diluted RT product may interfere with PCR reaction)

95°C for 10 minutes (Taq activation) 95°C for 5 sec

|

60 for 5 sec

|

72 (1sec/25bp to amplify)

|

x 40

70°C → 95°C with 0.1°C/sec ramping for melting curve

‡‡‡

LightCycler – FastStart DNA Master SYBR Green I, Roche - keep @ 4°C after first thawing and @ dark Should be optimized – start @ 4mM **** Should be optimized – range of concentration: 0.3 – 1µM §§§

Analysis of Nucleic Acids 13