Normalising RNA concentrations
Posted 01 June 2010 - 01:25 AM
86 ng/ul, 120 ng/ul etc.
I want about 2 ng/ul for the cDNA synthesis, so have to dilute them but how do I calculate how much water to add to get them all a 2 ng/ul.
Can I use C1V1=C2V2? I am so out of my depth with this as have barely done any molecular biology, can anybody explain this to me in simple terms ??
Posted 01 June 2010 - 03:00 AM
C1 (V1) / (V1 +V2) = 2 ng/ul;
where C1 is the current conc. in ng/ul, V1 is the vol you want to aliquot from the stock, (V1 + V2) is the final volume, V2 is the vol of sterile water you need to add and 2 ng/ul is the desired concentration.
E.g. Your stock conc is 120 ng/ul, and you want to dilute 1 ul of the cDNA to final concentration 2 ng/ul.
120 (1) / (1 + V2) = 2
120 = 2 + 2*V2
V2 = 59 ul
In other word, it's a 60 x dilution of your stock
Posted 01 June 2010 - 03:58 AM
Posted 01 June 2010 - 04:20 AM
The reason I am diluting so much is that I am using the Roche Transcriptor high Fidelity cDNA synthesis kit, the protocol for this specifies that I use 1 ng - 4 ug of total RNA in each cDNA reaction. The protocol says that the cDNA can then be used in PCR directly and suggests using 1-5ul of the cDNA reaction in a 20 or 50 ul final PCR product. I have never done this before so was just following the Roche protocol to the letter. Am I about to make another mistake???
Posted 01 June 2010 - 04:23 AM