Hey! I'm wondering how to calculate the amount of DNA templates to use in my PCR
For example, one of my samples has 359 ng/ul DNA (from NanoDrop)
For the PCR I want to use 40 ng DNA template.
The wells in my PCR will include in total a 10 ul of the following:
- 5 ul Sso
- 0.5 + 0.5 ul fw and rv primers
- 4 ul DNA template (with a conc of 40 ng DNA in it)
Do you happen to know how I calculate a final concentration of 40 ng DNA in this 4 ul template?
Thanks in advance.
if you have a concentration of 360ng/ul and need 40ng then dilute your dna by 360/40 or 1:9 then use iul of the diluted dna sample
Paul Rutland Thank you for your answer!
I forgot to mention in my question that I am using the SYBRgreen master mix which includes the Sso mix.
The wells in my PCR will include in total, 10 ul of the following:
5 ul Sso
0.5 + 0.5 ul fw and rv primers
4 ul DNA template
Do you happen to know how I calculate a final concentration of 40 ng DNA in this 4 ul template?
Best regards
Sunna
you want 1ul of 1:9 diluted dna to get 40ng so if you want to add 4ul of dna you should dilute the original dna to get 10ng/ul ( so a dilution of 1:36of the original) of original dna then add 4ul of this 1:36 dilution in your pcr reaction
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