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Single RE site cloning


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3 replies to this topic

#1 LacquerHead

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Posted 24 February 2013 - 07:42 PM

Hi,

Trying to clone an insert into a single BamHI site in my vector, wondering what the preferred method is here. I was going to cut the vector with BamHI in the presence of alkaline phosphatase, and then create BamHI sites on both ends of my insert which I will also cut with BamHI after PCR amplification, finally using this cut BamHI sticky end product to ligate into my vector. How does this sound? Any other suggestions?

Thanks!

#2 phage434

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Posted 24 February 2013 - 08:13 PM

Instead, design primers for your vector which amplify the vector with two different RE sites. Put similar sites on your insert primers. PCR both, purify, mix, cut with both enzymes and DpnI, ligate, go.

#3 LacquerHead

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Posted 24 February 2013 - 08:48 PM

Do you suggest first cutting the vector with the single RE before amplifying it with primers that have the new restriction sites?

#4 GNANA

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Posted 25 February 2013 - 05:12 AM

No, you dnt have to cut the vector before amplifying it.
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....




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