Standard Curve Protocol

Materials needed:

cDNAs
2X SYBR green master mix
Primers diluted to 10uM
RNase/DNase free water

Steps:

1.  Each cDNA reaction, described previously, should yield 20uL of product, with 1ug of added RNA per sample.

2.  Dilute the entire 20uL of your cDNA with 180uL of water.  This will reduce pipetting error later on. 

3.  For each primer you are testing, you will need 10uL of pooled and diluted cDNA.  Assemble 6 tubes, labeled 1 to 6.  For example, if  you are doing 10 new primers, add 110uL to tubes 1-6.  Add 110uL of your cDNA to the first tube.  Mix.  Take 110uL from the first tube, and mix with the second tube.  Mix.  Continue to the 5th tube.  Do not add DNA to the tube 6. 

4.  Add 10uL of cDNA for each of the standard curves to your plate. 

5.  Next, prepare the master mix following the recipe (see the excel spreadsheet), and pipet 15uL of the master mix to each well. 

6.  Each well should now have 25uL (10uL of cDNA, and 15uL of your master mix).