PCR products cloning

Did you add alkaline phosphatase after vector digestion. If you don't remove the phosphates the vector can re ligate on itself and this will interfere with successive ligation and result in the increased number of colonies.
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I have been having the same problem. Although I am not using the same sites (i'm using Kpn and EcoR1 sites) I am getting practically 0 colonies. I have yet to find a solution to the problem. The last solution I tried was to find someone that had successfully cloned a gene into pYES2 and cut it out and then subsequently place my gene into it. However, the transformation failed.
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Jon,

I have successfully used the Zymoclean Gel DNA recovery kit. It has worked pretty good for me every time I have used it.
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Hi, I have been trying to clone 520bp PCR fragment in the vector for months, after Ligation am getting lots of transformants but without my insert in it. Could you please help me with this problem.
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I have been trying to clone 300bp insert into pGEX vector with BamHI and Xho I site. I could't get the clone.Please help me with some strategies to cget positive clone.
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is it sure that the PCR products generated by Taq polymerase produces sticky ends. to get a blunt end what are the enzymes to be used?
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I am having problems in amplification of very small fragments <200bp, does someone has nay suggestion?
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hai everybody
proplem,
i have cloned my PCR product size of 700bp size by using pPROE. my restriction site is pst1 & PamH1. lication carried out by using T4 DNA ligase. i got clones with out my insert gene. what i do to get positive clones
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Hi everyone!

I have trouble cloning two PCR fragments of about 800bp, each one that are coming from a 5´RACE. I have done the same things that I did for the 3´RACE products, but I still have no success. I am using TOPO vector and I had to purified the products from gel, just like with 3´RACE products.
Thanks a lot!
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Anyone that can tell me how to overcome the problem of PCR fragments (between 200-500bp) not getting into vector (i.e. b-gal basic) is more than welcome!!!! I'm trying for months and even the zero-blunt Topo cloning seems not to work... Any suggestions from people with experience would be helpful.
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Iam trying to clone a 1.6kb plasmodium DNA in pGEX vector, after digesting with EcoRI and XhoI, I tried to ligate into pGEX and transform competent cells without success only once did I see a single colony grew on ampicillin plate. What could be the prob? any suggestions
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Hi!!

i am facing problem in cloning PCR generated fregment of 522bp, it is amplified with Deepvent polymerase(NEB), and have terminal sites(xhoI, KpnI), i am trying to clone in sk+, but not get success from months, even tryied for blunt end cloning but no success

please help

thanks in advance
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Hi,



I am having trouble inserting a second PCR product into a vector that already contains a promoter in the MCS. This is a cis element that I want to insert outside the MCS. Only trouble is, even after dephos. and linearization of the vector, it still seems to re-ligate time and time again. Any feedback would be helpful!
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Natalia:

Try TA cloning
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I amplify the TPA gene by using diagnostic primer but i am still unable to get the band of TPA gene by using the primers used for cloned them in pet vector

please guide me what will be the problem as i also do the gradient PCR for this gene
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hi all

I am having a problem in cloning of PCR product into the pBT2 vector the restriction sites included in the PCR product are BamHI and sphI

I am trying to digest and ligate into the pBT2 since several months
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hi all,

iam trying to clone two PCR fragments into PCDNA 3.1 vector cut with EcoR1 and Xho1 enxyme.Iam not getting any clones. Can u help me in this..........
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Hi! I'm trying to clone small inserts of DNA (50pb) digested with two different enzymes into the pGEX vector. I purified my digested fragment from PAGE 8%. I then ligated it to the digested vector but i don't obtain any transformants.



Has anyone cloned such small fragments ??

Thanks
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Hi! I'm trying to clone cDNA into TOPO vectors, but it seems that the DNA degrades during or after purification from agarose gels.

If someone could give an advice it would be good!




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Hi to U all,

I've been trying to clone a 2,5 kb fragment into a pHannibal which has been double digested by Xho1 and Kpn1? The problem is that I am not getting any transformants.

I request for a help to enableme undersatnd what is happeneing.




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Dear friends,

I have a problem. I am trying to insert a 2.1kb gene into pYES2 (5,9kb) using XhoI and BamHI. The efficiency of tranformation is almost 0. (I have maximum 8 clones in 4 agar-plates) Any of you have any idea why?

kind regards, Momeu
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Hiya all. I've been trying to clone a PCR fragment into a vector which has been double digested. The vector size is 10kb and the insert size is 0.35kb. The problem is that I am not getting any transformants, though the control (single digested DNA of both enzymes, self ligated) are showing good efficiencies of transformation. Also the double digested vector, which has been subjected to self ligation is not showing any transformants (as expected). How do I increase the efficiency of transformation?
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Dear Friends,

I am cloning PCR product in pGEX-4T-1/BamHI/XhoI (insert size is 1.1 kb)


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Dear Friends,

I am cloning PCR product in pGEX-4T-1/BamHI/XhoI (insert size is 1.1 kb)


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I am looking for bacterial plasmid pZML793 which used to clone adh gene in maize,I don't know who and where have it ? I request for a help that a ncie man would enjoy tell it to me!


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HI to you all;

Problem.

I have cloned my gene in pmosBlue vector, however i need to confirm that the gene is their by restriction digest. I have the restriction map and chose my enzymes carefully those that don't appear in the insert. However on restricting i get 2 band one of which is of the size of my insert, the other band is twice the insert size. And in addition i get the size of the backbone vector of the expeted size. Now my worry is this extra band, What could be the explanation?



I request for a help to enableme undersatnd what is happeneing.





Regards

Eddie
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cloning the pcaA gene of m.tuberculosis


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I have trouble getting DNA out of the PAGE gel.

I have tried the Qiagen kit, elute gel slices with Sodim acetate at 50 degree over night.....none of the techniques worked....
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I have trouble cloning my PCR fragments. These fragments come from an amplification that gives lots of bands but I have to clone the bands between 300 and 1000 bp. So I have to cut the gel and make an extraction. My PCR is performed with Taq and I have tried TA cloning, as well as blunt cloning after treating the PCR products with T4 DNA polymerase and pBlueScript (blunt cut) with alkaline phospatase. None of the techniques worked...
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Try cutting the pET with a blunt cutter, SmaI or EcoRV are in a lot of MCS, then add the PCR product (blunt it first, or amplify with a polymerase that leaves blunt ends - not Taq). When you come to do the ligation, add just a little of the same enzyme...it should elimate negatives.



Jon

webmaster for http://molbiol.net/


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