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Difficulties in cloning long PCR product
Posted by: rajugottumukkala (IP Hidden, New member, 6)
Date: May 18, 2006 12:57PM
Hi All,
I have been trying to clone 6kb long PCR product into pCMVTNT vector of 4kb (Promega) at EcoRI sites but no success for a long time. I amplified PCR product with EcoRI ends in the primers from RT-PCR product. When I ran on 1% gel I do see 6kb product when compared to the marker. Then I purifiy the PCR product after running on 1% agaroge gel using Qiagen agarose gel purification kit. Subsequently, I digest the PCR product and vector with EcoRI enzyme (NEB) for 2 hrs at 37C. I do dephosphorylate the vector using antarctic phosphatase (NEB) for 1 hr at 37C. Later, I purify both digested PCR product and dephosphorylated vector using Qiagen PCR purification kit. Thenc I run the purified products on agarose gel before ligation. Once confirmed then I set up ligation at different ratios (1:1, 1:3, 1:5 and 3:1). I do include digested vector alone, digested and dephosporylated vector alone in ligation. All ligations were done at 16C overnight using T4 DNA ligase (NEB). Next day I did transformation (Top10 chemically comp cells) according to the Invotrogen protocols and plate 100ul of culture on agar plates and incubate overnight at 37C. On the next I see lawn of coonies on the plate with digested vector alone. I do see colonies on dephsophorylated vector alone plate but these are very less compared to the digested vector alone plate. In addition, I do see the same number of colonies on plates with vector+insert (at different ratio) similar to dephosphorylated vector alone. There is no difference between them. When I screen 30 clonies from vector+Insert plate with restriction digestion I do not see at all any sample with insert. Why???? There are two funny things are happening in experiments. One is why do I get colonies on dephosphorylated vector plate alone??? though I use antarctic phosphatase, which is very good for removing phosphate gropus compared to calf intestinal phosphatse. why do I get the same no of colonies on dephosphorylated vector plate and vector+insert plates? Secondly, why none of colonies has inserts? If any body has answers please let me know. Any suggestions are appreciated. Thanks Raju
Re: Difficulties in cloning long PCR product
Posted by: mitolab (IP Hidden, Senior member, 89)
Date: May 19, 2006 07:56AM
It seems to me the dephosphorylation is incomplete. You can try dephosphorylation again using different phosphatase. Or better off designing new primer pairs with two different cut site for directional cloning so you can avoid dephosphorylation step. Another common cause is your PCR product wasn't digested efficiently. Did you include 2-4 extra nt after your EcoRI sequence in your primers?
Re: Difficulties in cloning long PCR product
Posted by: rajugottumukkala (IP Hidden, New member, 6)
Date: May 19, 2006 01:41PM
Hi,
Thanks for your suggestions. Regarding dephosphorylation you might be right. I haave to try a different phosphatase from a different company. Since Antarctic phosphatase (AP) is better than calf intestinal phosphatase (CIP) according to NEB, I used 2ul of AP for 2ug DNA and incubated for 30 min at 37C. Again I added 2ul of AP to the reaction and continued for 30 min more at 37C. With respect to directional cloning, initially I tried to do this with EcoRI and XhoI sites but no success (I thought problem was due to double digestion though I followed according to NEB suggestions) so changed to clone using only one restriction site. I included 2 extra base pairs of nucleotides after restriction sites since my PCR product gives an extra of A hang overs. Total it would be at least 4 base pairs. The other thing you mentioned was undigestion of PCR product. It could be one of the reasons. It is hard to determine on the agarose gel whether it has been digested completely or not. Is there any way to determine the digestion of PCR product? In the mean time I am trying with new primers with extra 10 bases after EcoRI site. Let's see what will happen. If have any ideas please let me know. Thanks in advance. bye Raju
Re: Difficulties in cloning long PCR product
Posted by: centromere2005 (IP Hidden, New member, 1)
Date: June 1, 2006 03:55PM
To me, if the number of colonies on dephosphorylated vector plate = vector+insert plate, that means incompleted dephosphorylation.
If there are no inserts but the digested vector does have many colonies, that means incompleted digestion of PCR product. It happens occationallywhen you digest using PCR product directly, no matter you did gel purification or you do have protective bases. You can order new primers or the better way is to ligate you PCR product into a T-vector, then cut it from the plasmid.
Re: Difficulties in cloning long PCR product
Posted by: kambakamsekhar (IP Hidden, New member, 1)
Date: June 6, 2006 03:31AM
hello raju,
ur 6kb PCR product, 1st cloned into TA cloning vector then ligate into other vector in whatever interst site. anther option is u applified ur PCR product using pfu enzyme instead of tag,these product u directly ligate into any bluntend site avaible vectors.
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