Hello,
I'm validating the primers for GAPDH, Tubulin and HSP70 with 8-fold serial dilution (1: 2) and the efficiency is very high and with points outside the standard curve. I read about reagent contamination and changed everything. Other people in my lab have already been able to validate some these primers in other tissues.
What could be happening?
Make sure when you are doing your serial dilutions that the DNA concentration in your dilution stays the same. I would suggest adding some nonspecific chromosomal DNA, like salmon sperm DNA. If you just dilute your target, without a carrier DNA you never will get a nice tight standard curve.
Good luck,
Alex Ryncarz PhD
What primer concentrations are you using? Is the Tm standardised? The dilutions you mentioned are for the template I suppose. How many dilutions did you use ?
Hi Jaquelene
In which you diluted primer? TE buffer or NFW?
And how old your stock primers are ?
If primers are too old then many times it will not give same results as previous.
Thanks all!
I solved diluting my cDNA for 1000 ng and everything worked.
when you are doing your serial weakenings that the DNA focus in your weakening remains the same. I would recommend including some nonspecific chromosomal DNA, similar to salmon sperm DNA. On the off chance that you simply weaken your objective, without a transporter DNA you never will get a pleasant tight standard bend
regards
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