REAL-TIME PCR Protocol
Reagents: (volume/sample)
Rnase free water
*Q2X buffer
7ul
10ul
Primers (5 μM)
Total volume
1ul each
19 μL
* Sybergreen mix that contains dNTP, TAQ, MgCl2, Buffer
Note: Keep samples on ice throughout experiment.
Protocol:
1. Remove cDNA samples from -20C freezer and take out all necessary reagents.
2. Prepare and label tubes.
3. Prepare master mix. (prepare enough mix for all samples + 1).
N.B. Samples include samples + negative + standards**
** 4 different concentrations (e.g. 10-4, 10-5, 10-6, 10-7)
4. Distribute 19ul of mix to each tube.
5. Add 1ul of sample to appropriate tubes and mix well.
6. Add samples to capillaries within the cooling block. (volume of each sample = 20ul).
7. Prepare standards in another room (add 1ul of each standard) and add to
capillaries.
8. Spin down samples in carousel and put the samples in the lightcycler.