I will be performing both Sanger sequencing and RT-qpcr for my target gene. For Sanger, I have ordered 3 sets of primers (around 2500, full length). Is it possible to use the same primers for RT-qpcr as well or do I need another specific set of primers?
Yes, you can use the same primers of qPCR for sanger. be aware that you need to run each forward and reverse in separate reactions and you need further dilution for primer as recommended by the manufacturer. SORRY MAY BE I MISUNDERSTOOD your question***. If you mean that you are willing to use Sanger primers For Real-time PCR, then no . You need to shorter specific primers with 150 product length to be very effective. However,if you willing to use primers of qPCR for sanger, then it is ok as I have mentioned above.
No, you probably CAN'T use the primers you designed to sequence a 2500bp region for qPCR.
For one, unless there is a ~2400bp intron in that region, your product is way, way too big to amplify efficiently in qPCR.
Primers for Sanger can be in any region of the DNA, primers for qPCR must be in an exon or UTR.
You'll most likely need to design and test several pairs of primers to find ones that have 95-105% efficiency in qPCR.
Good luck!
Hello! thanks for replying. Actually for sanger, I want the entire transcript to be sequenced and for qpcr I have identified domains which are around 200 to 250 bps.
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