I am going to dispatch PCR amplicon of my plants obtained from the rbcL primers for sequencing. Someone told me that it will not only need the Rev: & Fwd: primers but will also need a third internal primer for sequencing. Can anyone please help me in finding the sequence of that internal primer to be needed in sequencing?
thanks
It depends on how long the amplimer is and how much of the amplimer sequence that you need. You can sequence 800-900 bases in each direction and you will not get any useful sequence under each primer or for the next 30 bases from the primer. So If your sequence is 600 bases then you do not need any other primers because each primer will sequence the full length up to the end of the other primer. If your sequence is 1200 bases then you will only get single sequencing product for about 400 bases at each end and good double sequence only in the middle bases. If your primers are based 60 bases into an intron and you only need exonic sequence then not having double stranded sequences at each end will not worry you. If your primer is 60 bases from the intron and the whole amplimer is less than 900 bases you may choose to sequence in one direction only to save money and just sequence from the other direction any samples that look interesting or sequence poorly
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