Sequencing Errors

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sagarvarankar's picture
Sequencing Errors

Hello everyone..

Recently I finished some cloning work in my lab and prior to setting up my actual experiments wished to confirm the constructs generated. The constructs validated out in PCR and RE digestion, however, sequencing is not yielding any results. I wish to ask does the use of a specific primer dictate the sequencing chemistry, as I have not used the vector specific sequencing primers but have used the insert specific cloning primer instead. Would this have a great impact on sequencing as I already have seqyuencing plates full of no results i.e. no sequence.
Could someone kindly inform me if the seqneuncing primer is absolutely required as opposed to the cloning primer.

Nikail Collins
Nikail Collins's picture
sequence clone

Hi,When I sequence the purified product from a plasmid insert, I use multiple primers including the start of the insert (cloning primer) to ensure I am able to get all the overlap necessary to provide entire target sequence. It also helps in making sure your sequence is going in the correct direction.