I have extracted DNA from flies. I quantified DNA using nanodrop and got concentrations ranging between 1 and 30 n/ul of most of the samples. For some, concentrations were much higher: 14000, 1262, 469, and 520 n/ul.
For extracts less then 5 n/ul, would it be difficult to get a successful PCR reaction?
I belive that the two first concentrations should definitely be diluted for PCR but how about the 2nd and the 3rd ones?
How do I make the ditlutions and to which concentrions should I dilute them?
1) You can dilute the DNA using M1V1=M2V2 formula using either TE buffer or sterilized ultrapure water
2) Amount of DNA template per reaction depends on the polymerase that you use, normally 50ng/reaction is fine
3) If you are using 50ng/reaction, you can dilute DNA to 50ng/ul for easy calculation during PCR
4) For DNA less that 5ng/ul, u can always try to increase the dna template per reaction
Hope this helps!
Yes agree with Vikki Wong, template can be diluted using this formula M1V1=M2V2,
But too high concentrations from samples of same volume may indicate impurities, look for 260/280 ratio if it is fine then dilute your template with ddH2O according to the formula,
Best of luck
Just a quick suggestion, try to run your DNA samples on agarose gel once before proceeding to PCR reaction.
I usually use 3 ng of DNA diluited in 10 uL/EB (3 nanogram of DNA into 10 microliter of diluition buffer , that generally is TRIS).
Firstly, it is important the DNA concentration that you have to calculated in cellular extracted DNA).
Best regards,
Carla MICHELI
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