standard curve protocol
The standard curve provides you with information about each run. First, it lets you know that your amplification is working. Second, it allows you to calculate the exact amplification efficiency, rather than assuming 100% efficiency of amplification.
- cDNA prepared from your RNA
- 2X SYBR green master mix
- Primers diluted to 10 uM
- RNase/DNase free water
1. Each cDNA reaction, described previously, will yield 20 uL of product, which originally had 1 ug of RNA per sample (or so).
2. Dilute the entire 20 uL cDNA reaction 1:10 by adding 180 uL of water.
3. For each primer you are testing, you will need 20 uL of pooled and diluted cDNA. Assemble 6 tubes, labeled 1-6. For example, if you are testing 10 new primers, add 110 uL of water to tubes 2-6. Add 220 uL of your mixed CDNA to tube 1. Take 110 uL of this mix to tube 2. Mix, and take 110 from tube 2 to tube 3. Continue like this until you get to tube 5. Do not add any DNA to tube 6.
4. Add 10 uL of cDNA for each of the standard curves on your plate from the mix.
5. Prepare a master mix following the recipe found on the excel template and pipet 15 uL of that mix to each well. You will make a different master mix for each of your primers.
6. Each well shoudl now have 25 uL (10 uL of cDNA, and 15 uL of your master mix).