I have a multiplex PCR with 6 Fwd species specific primers and one universal reverse primer. I usually run single PCRs and after this I clean them with exosap, then cycle sequence each with each primer and then do an ethanol precipitation before sequencing in an abi3730. Do I need to add M13 tails? Or how would you think it could work the best?
Not a terribly good idea to sequence after multiplex PCR. But you can try it.
In the Sanger sequencing reaction, you need to specifically sequence only one product. You cannot "multiplex" Sanger sequencing. So naturally, you cannot use the reverse primer as a sequencing primer since that would sequence up to 6 products simultaneously. Different tails will not change this and will therefore not help.
You could use one of the forward primers each in your sequencing reaction. If your six forward primers are indeed specific, that should give you specific sanger reads.
Of course, if your 6 products are of different size you could purify the products on a gel, as suggested by Angus. After that, you could also sequence using the reverse primer. But, quite frankly, I would just set up six seperate PCRs, which is probably less work and not more costly. Plus more likely to work than anything else.
I would just run the products out on an agarose gel, then excise and purify DNA from each of the bands I want to sequence.
It might be easier to just run each PCR singly (only include one forward primer in each of 6 reactions).
You could also just sequence using only the forward primers.
Not a terribly good idea to sequence after multiplex PCR. But you can try it.
In the Sanger sequencing reaction, you need to specifically sequence only one product. You cannot "multiplex" Sanger sequencing. So naturally, you cannot use the reverse primer as a sequencing primer since that would sequence up to 6 products simultaneously. Different tails will not change this and will therefore not help.
You could use one of the forward primers each in your sequencing reaction. If your six forward primers are indeed specific, that should give you specific sanger reads.
Of course, if your 6 products are of different size you could purify the products on a gel, as suggested by Angus. After that, you could also sequence using the reverse primer. But, quite frankly, I would just set up six seperate PCRs, which is probably less work and not more costly. Plus more likely to work than anything else.
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