can anyone tell me what is the difference between hotstar master mix and normal master mix?
I am dealing with FFPE DNA and facing problem ; bands are not coming consistent
should I design my primers in such a way as amplicon size is less than 250bp?
what protocol should I follow for doing PCR as my downstream application is sanger sequencing...
thank you
Yes it is very much possible.
While dealing with FFPE DNA you have to take extra care while designing primers otherwise there are more chances that PCR will not work.
- Never design primers for amplicons bigger than 250 bp. 200 bp is ideal length.
- Keep the annealing temp in the range of 58 to 62C.
- Do the optimization of primers by setting up a PCR at at least three different annealing temperatures.
I hope taking extra care with the primer design and setting up a clean reaction will work.
best of luck !
thanks Kaleem Iqbal
but if i design amplicon 200bp then it will be difficult to be detected in sanger sequencing as in sanger sequencing firt 50-100 bp nd last 50-100bp are nosiy data...
can you please clear my doubts
Thats right! you can leave 50 basepairs upstream and downstream. In this case if your target amplicon will be reduced to 150 readable base pairs.
If your desired length is more than 150 then you can split the target in two parts and design overlapping primers. e.g. if your target amplicon is 300, you can split this in two parts of 150 each and design primers surrouding this region--not within this region--.
I have made a picture, Hopefully it would help. See the attachment please.
thanks a lot Kaleem Iqbal
i did'nt thought in this direction
it's very very helpful
I am glad that it helped. Kindly upvote the answer that you find good. Thanks :)