I am trying to perform sequencing of different antibiotic genes carrying plasmid. I have amplified the respective genes separately with pcr and now want to sequence them.
Can anyone suggest me which primers should i use for sequencing? Also i want to sequence them in Macrogen and they offer a lot of sequencing primers.
Hi there,
You can use the primer set you have used for amplification but you will have to provide them or to make them synthesized by the company. But if the gene to be sequenced has been cloned between a set of sequencing primers you may use those. Bear in mind that sequencing starts to be efficient around 50 nucleotides downstream the primer.
You can clone your PCR products into pGEM-T or other plamids containing T- over hangs (if your DNA polymerase adds A-overhangs). Then you can use some internal primers ( eg M13) to sequence and you will not lose any bases from the beginning of your construct.
Or would not it be possible to sequence directly your original plasmids?
Use the same primers you used to amplify the sequences. Make sure the samples are free of all primers (do a clean-up with any of the existing silica-based PCR purification kits) before you ship them, or you will get mixed sense/antisense base calls in the same sequencing run. You could also inquire directly with Macrogen, I think they can do the purification for you if needed. And remember to sequence both strands, since the reads will be missing the sequence of the primer itself and the first 30-50 bp downstream.
The primers offered by Macrogen bind to sequences flanking the cloning sites of well-known cloning/expression vectors. If you know for sure that your amplicon contains them you can use them; otherwise you´d have to clone the PCR products into pGEM-T or something similar.
Thank you all for the response. I am not thinking of cloning the genes currently. I am trying to just perform sequencing in Macrogen. They offer a lot of variation in primer and upon exploration i found that T7 FORWARD AND M13 reverse is most commonly used.
I would like to know are these primer good for sequencing?
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