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Problem cloning PCR product into double-cut vector


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#1 biomol

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Posted 25 October 2004 - 06:48 AM

I am trying to clone a PCR product with sticky ends Bgl2 / Hind 3 into a commercially bought vector, namely pEGFP1 bought from Clontech. I have performed nested PCR and used primers with the above enzyme sites at the ends of the foward and reverse primers. After purifying the PCR product using the gel and Qiaex gel purification kit , I digested both the PCR product as well as the vector with the above enzymes then dephosphorylated the vector with CIP and purified both again using the gel. After quantifying the purified products .......I did the ligation using T4DNA ligase from Promega at 1:3, 1:5, 1:10 ratios of vector to insert . I am not getting any clones after transforming DH5alpha ........and if there are any clones they appear after 30 hrs but have no inserts. Did minipreps and then digested the minipreps with the enzymes above.

Could someone tell me what is wrong ? Does anyone have experience working with the Clontech vector pEGFP1 ? Thank you !

#2 biomed

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Posted 26 October 2004 - 08:36 AM

Check your PCR primers first. Do you have enough bases after enzyme site? It is more possible you did not digest your PCR product.

#3 biomol

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Posted 27 October 2004 - 06:05 AM

Check your PCR primers first. Do you have enough bases after enzyme site? It is more possible you did not digest your PCR product.

Hello biomed
I have two bases after the enzyme sites. I have just cloned in my PCR product into pCR2.1 vector by TA cloning . And this after having used PFU polymerase and then adding the A overhangs with Taq incubation . Transformed and got colonies .......... did minipreps digested with the same enzymes and got back my insert intact. I now have doubts about the Clontech vector which I have been using .pEGFP1 .Does anyone have experience using it ?

#4 biomed

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Posted 27 October 2004 - 11:33 AM

Check your PCR primers first. Do you have enough bases after enzyme site? It is more possible you did not digest your PCR product.

Hello biomed
I have two bases after the enzyme sites. I have just cloned in my PCR product into pCR2.1 vector by TA cloning . And this after having used PFU polymerase and then adding the A overhangs with Taq incubation . Transformed and got colonies .......... did minipreps digested with the same enzymes and got back my insert intact. I now have doubts about the Clontech vector which I have been using .pEGFP1 .Does anyone have experience using it ?

Hi,
BgI I requires at least 2 bases at the end, but Hind III requires at least 3 bases.
I used pEGFP-N3 and did not have any problem.
Good luck.
Biomed




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