Dear all,
I'm learning and taking the same genotyping PCR experiments and want to get / share some advice or experience on obtaining the right bands for the data.
I ran 3 samples of Stra8-iCre and 2 out 3 samples were not wild type, which was different genotype compared to the actual data from our laboratory and all of them were supposed to be wild type.
There was no contamination nor mistakes on performance of the protocol and I also regulated annealing temperature into 58 or 60 degrees Celcius for this whole times.
I wonder how can I get the right bands for Stra8-iCre and what was I missing except for the accuracy of the protocol and regulation of the temperature.
Hi Dayun Lee
choose an annealing emperature that does amplify and run a pcr set with 0. 1%, 2% up to 8%dmso and at some dmso concentration the pcr will probably be a clean band. If your gene has pseudogenes or is one of a family of similar genes then touchdown pcr or new primers might be needed.. You can perform primer gradient PCR to optimize the concentration of primers. Also, You can use pico molar concentration of primers. or use different gradient pcr such as with annealing and denaturation etc.
Elham Yaghoubi
Dear Professor. Yaghoubi,
First, I genuinely appreciate with your reply and the time you put into this inquiry.
And I calculated the concentration of the primer and asked DMSO
and some other details for the experiments and hopefully, it went
correct. I am genuinely thankful for your kind devotion.
Thank you.
The non-specific binding can be overcome by optimizing the annealing temperature for your primers. Try the gradient PCR with a temperature range of -+2 degrees for the temperature of your primers. You might see that at a certain temperature you will get a distinct band and choose that temperature for your next reaction, and you will get only the target band.
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