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sequencing slightly degraded DNA
Hello,
If one amplifies CO1 in two parts with interior primers. Would it be possible to sequence the two parts together in one step?
I am asking because we assembling a plate with PCR products of 74 good DNA, but in 20 other samples the DNA is not so good, and we are going to try to do barcodes in two fragments, and I wonder if I could put the two fragments in the same well to send them to be sequenced in one 94 well plate. I know it might be risky, but maybe some of you have tried this.
Best, Patricia
If one amplifies CO1 in two parts with interior primers. Would it be possible to sequence the two parts together in one step?
I am asking because we assembling a plate with PCR products of 74 good DNA, but in 20 other samples the DNA is not so good, and we are going to try to do barcodes in two fragments, and I wonder if I could put the two fragments in the same well to send them to be sequenced in one 94 well plate. I know it might be risky, but maybe some of you have tried this.
Best, Patricia
Views: 10
Replies to This Discussion
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Permalink Reply by Lee Weigt on -
I don't think so -
unless you only wanted the original terminal primer sequences - resulting in a single strand read (with a possible gap in the middle).
I'd do each half-amp independently with the 2 primers used for the amp - maximizes information return, and we are only talking about 40 extra sequencing reactions in this case.
Lee -
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Permalink Reply by Patricia Escalante on -
Thanks you.
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Permalink Reply by Kris Jett on -
Hi Patricia,
You might also want to post this question to the Lab Procedures discussion board. This way more network members will see it, increasing the chances you'll get helpful tips and suggestions. -
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