I know for doing sequencing, only one of forward or reverse primers are used separately.
I want know if sequencing can be done in this way.
if they were mixed together, the result of sanger sequncing will be messed up...
You can't. You will obtain mixed results and cannot read your sequences
Hi Mahmoud, The Sanger protocol recommends creating a mastermix of the foward and reverse primer separately. After which you set up a forward reaction with your template in separate tubes, and do the same for the reverse reaction. This enables you to create both F and R fragments separately where for example each sample will eventually have a F sequence and a R sequence. This in a way serves as a quality control procedure. If you try mixing the primers up in one reaction tube, you'll end up with too much noise, characterized by trace files with overlapping peaks which cannot be called to bases by the basecaller.
In order to obtain proper sequences, you cannot mix your primers...
Practical answer is NO.
Theoretically you might have Sanger sequencing with one primer labelled with P32, P33 or s35 or any other isotope, and the other primer labelled with biotin. Then separated on PAAGE and blotted on membrane and processed the two ways - exposure on film, and chemiluminescence
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