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y pGEMT first then pGEX-2T


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#1 eclatant

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Posted 04 March 2009 - 01:21 PM

Hi guys, i'm pretty new to molecular biology and there are some things which i don't get in directional cloning. Why do we have to ligate my pcr product into pGEMT vector first, express it in e coli, then take it out again and ligate into the pGEX-2T vector. This will give me the plasmid with my DNA and GST.
Why cant i just ligate the dna product directly into the pGEX-2T Vector?

Thanks

#2 Heir Stylist

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Posted 13 March 2009 - 08:42 AM

Hi guys, i'm pretty new to molecular biology and there are some things which i don't get in directional cloning. Why do we have to ligate my pcr product into pGEMT vector first, express it in e coli, then take it out again and ligate into the pGEX-2T vector. This will give me the plasmid with my DNA and GST.
Why cant i just ligate the dna product directly into the pGEX-2T Vector?

Thanks


If your PCR product have the restriction sites for cloning, you can skip the T/A cloning procedure ( cloning into pGEM T easy vector). Just digest the PCR product with the selected restriction enzymes and ligate into the pGEX-2T vector digested with the same enzymes.

Regards
aviprem

http://aviprem.gq.nu

#3 tyrael

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Posted 17 March 2009 - 12:33 AM

Hi there.

if my purpose is to send for sequencing,
after i get my PCR product,
i will just have to ligate it into pGEMT-Easy vector (is it so called as T/A Cloning since i m using the extra 3'A tail of my PCR product and ligate into the T-overhang in vector? ) and then transform the vector into bacteria (E.coli) ?

due to my purpose is just for sequencing, so it is not necessary for me to restrict my gene right ? (some more i don know my gene sequence...)




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