QRT-PCR

2/4/04

 

Part 3: Quantitative PCR

 

 

 

  1. Using SYBR green kit from ABR (cat# ). Sign up for the machine, and start warming up the lamp when you begin to prepare the mix.

 

  1. Prepare master mix for about 1.35X the number of final wells

 

                                          22uL total per reaction

2.5uL 10X PCR reaction

3uL MgCl2

2uL dNTPs

0.125uL enzyme

                                                14.375uL ddH2O

 

  1. Mix well after each mix, and then spin down. Divide master mix into separate tubes for each of the conditions (for example if -, KCl, cAMP then make 3 tubes). Into each put enough for 8 wells (so 176uL). For the standards (8-12 total) you will need only enough for 3.5 wells (77uL) since these will not be split for two sets of primers.

 

  1. For each primer pair, run at least 3-5 standards and a zero. For the standards add purified quantitated PCR products that are stored at 1ng/uL. Doing 10 fold dilutions, for GAPDH use 10-3 to 10-7 (if <5 go on the higher side), for 18S 10-2 to 10-6 (if <5 go on the higher side) and for BDNF or CaRF use 10-4 to 10-8 (if <5 go on the lower side for CaRF, higher for BDNF). For the zero, add ddH2O rather than PCR product.

 

  1. Add 1uL cDNA/well (so 8 in the current scheme for samples and 3.5 for standards.)

 

  1. Split mastermix/cDNA into two tubes, each with 3.5 wells worth of mix (so 3.5 x  23 = 80.5uL). Standards do not need to be split.

 

  1. Add 1uL each primer per well (so 3.5uL forward and 3.5uL reverse to each tube). Pipette 25uL in triplicate from each tube into the assay plate.

 

  1. Document which samples are in which wells.

 

  1. Seal the top of the plate with a plastic sticky cover. Stick down hard with the roller. Do not touch the plastic with your hands. Spin down the samples for 1Õ in the tc centrifuge at about 1000RPM.

 

  1.  Run program.

 

  1.  Primers: CaRF RT5A-RTshort. mBDNF BdnfF-BdnfR2.  GAPDH mGapdhF-mGapdhR,  18S m18SF-m18SR. Stocks 1ug/uL, dilute 1:10 for working stock.