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Building cDNA constructs to pcDNA3.1 directional TOPO vector


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#1 Neurochick

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Posted 17 November 2013 - 06:32 AM

Hi,

 

I am pretty new at this, I am trying to order my gene of interest, full cDNA construct from Genscript, and trying to ligate that to pcDNA3.1 directional V5-His-Topo vector from invitrogen. This is to simplify matters and not having to do the PCR/cloning for the construct. Genscript is placing the insert in puc57 vector. But the pcDNA3.1 directional TOPO vector kit includes the PCR step for which to enable directional cloning, the forward PCR primer must contain the sequence, CACC, at the 5′ end of the primer. The 4 nucleotides, CACC, base pair with the overhang sequence, GTGG, in pcDNA™3.1D/V5-His-TOPO®.(see attachments)

 

So I am unsure on whether I need to add the CACC to my ordered gene construct?

If its placed in puc57 vector and I have to digest using restriction enzyme to isolate the insert for ligation, won't the ends be different from CACC required and cause the ligation to directional TOPO to not work?

Please help, will appreciate any advice. Thank you

 

 

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#2 leyshmanioz

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Posted 17 November 2013 - 09:01 AM

The easiest way is to give the vector to Genscript, they do cloning into custom vector if you pay for it, i dont think its that expensive, otherwise you would have to perform PCR if the kit requires you to have those additional nucleotides at the end, but then you would have to sequence it.

I suggest to go with custom vector cloning considering you have very nice MCS there



#3 bob1

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Posted 17 November 2013 - 11:36 AM

The reason they say to do a PCR for the cloning is so that the overhang can be generated - it is incorporated into the primer, and the PCR won't fill it in.  If you get Genscript to make the sequence with the CACC included then it will be double stranded in the vector(unless you can find some way of digesting the sequence to recover the overhang - you would need to find a restriction enzyme that produces this site - try here), which will not allow you to do the directional cloning as you want.

 

Long story short - don't include the CACC in the sequence you are giving to Genscript.



#4 Neurochick

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Posted 17 November 2013 - 07:54 PM

Thats exactly what I was thinking and I have not yet found any restriction enzymes that fits the CACC. From the map attached, it seems that the insert sequence with CACC included will be double stranded in the vector, the complementary GTGG from the insert seems to be somehow displaced by the Topoisomerase to the pcDNA3.1 vector during the ligation.

 

So basically it cannot be done unless Genscript does a custom vector, am I right?



#5 bob1

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Posted 18 November 2013 - 12:28 AM

Correct






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