Real time PCR and RT-PCR reactions

Preparations

Prior to setting up real time PCR, you will need the following:

1. Reagents and disposables from ABI including a real time PCR kit for either Sybr Green or Taqman. We use the Taqman universal PCR master mix about $2/ 50 ul reaction) which comes with 2x master mix to which you need only add DNA/RT, primers and probes.

Sybr green PCR reaction kit ABI 4309155
Taqman universal PCR master mix ABI 4304437
Optical tubes (box of 2000 individual tubes) ABI N8010933
Optical caps (8caps/strip, 300 strips/box) ABI 4323032
96 well tube retainer (20 assemblies) ABI 403083

2. Primers (Sybr green and Taqman) and probes (Taqman only). Probes should be 5' labeled with 6'FAM and 3' labeled with TAMRA. If ordering from Qiagen, they should automatically be HPLC purified

3. DNA or RT reactions (see RT protocol in DNA/RNA section of general protocols).

Setting up reactions

(The following reaction description will talk in terms of quantifying different genes relative to each other as for a gene expression study. However, the same description applies to species surveys where you want to discriminate different species using the same (or different) gene. In this case, gene in the following discussion refers to species.)

In setting up reactions it is important to use master mixes as much as possible. This reduces pipetting error (note: the Sybr green and Taqman master mixes do contain a ROX normalization dye which does correct somewhat for pipetting differences between the wells). The GeneAmp 5700 is a single color machine. For each gene that you want to detect, you must set up a separate PCR reaction. This involves making a master mix for all the genes from a single sample that you wish to compare and then aliquoting them to separate tubes. You then make up a master mix of primers (or primer/probes for Taqman) and add then. It is useful to set up a grid with samples going across the rows and genes going down the columns (see powerpoint presentation for an example).

The first time you test your primer/probes, it is useful to do a negative control (water) and then a dilution series of your positive control. This will confirm that you do not have contamination and that your are working in the exponential regime where critical cycle number is linear with template concentration.

The following is a sample set up for a Taqman study of five opsin genes. This might be a test case where the individual samples would be a dilution series of seven different concentrations (1x, 0.5x, 0.1x, 0.05x, 0.01x, 0.005x, 0.001x) to test linearity. A dilution series is handy because it also can give you information on the PCR efficiency of each gene (see data analysis).

For each of the 7 RT samples (concentrations), make up the following master mix:
 Reagent For 1 rxn For 6 genes
2x Taqman mix 15 ul 90 ul
water  5.5 ul 33
RT reaction  0.5 ul 3 ul
Total 21 ul 126 ul
Mix well and spin. Add 21 ul to each tube in sample row.
For each of the five genes, make up the following master mix:
 Reagent For 1 rxn For 8 RT samples
Forward primer (3 uM) 3 ul 24 ul
Reverse primer (3 uM) 3 ul 24 ul
Probe (2 uM) 3 ul 24 ul
Total 9 ul 72 ul
Mix well and spin. Add 9 ul to each tube in gene column and pipette to mix.

Once the RT master mixes and primer/probe mixes are distributed, put on the optical caps (it is handy to do this for each column after you add the gene specific primer/probe mix-this reduces contamination and mispipetting). Spin down the plate. You are ready to run the GeneAmp 5700.


07/04 KC