PCR product cloning - (Jan/10/2008 )
Hi, is there anybody who can help me? I am doing the PCR product cloning. I need to assemble an PCR product of 5.5kb into a large vector of 11kb. Before, I have succeeded in ligation of 4.2kb insert into a 7.5kb vector. But now I just failed several times to do the sticky ends ligation of the 5.5kb PCR product into the 11kb vector. Control samples showed everything is no problem but I failed to ligate this large insert into the large vector for over a months experiment.
Is there anyone who have experience in ligation of the large insert into a large vector? Thank you.
Is there anyone who have experience in ligation of the large insert into a large vector? Thank you.
hi,
Mainly in such situations, the problem comes from the pcr insert digestion. i dont know what kind of controls you keep to ensure that ur pcr terminus is efficiently cut...
1) if your primers bear restriction site, ensure if they also provide sufficient extra overhangs that can support effective restriction digestion at the pcr product terminus. Ensure optimal digestion conditions for the restriction enzymes used to cut the pcr insert. you can increase the enzyme concentration and/or incubation time (with permissible final glycerol concentration to prevent any star activity ). check optimal buffer for double digestion conditions or try sequential digestion to ensure both the termnus is cut efficiently. For optimal overhangs for selected enzymes, go through
http://www.embl.de/ExternalInfo/protein_un.../extensions.pdf
2) you could also try reduced vector concentration, and/or final vector/insert concentration that helps in circularization of your 11kb vector to meet with the insert at its ends. 3) increase ligation incubation time 4) heat inactivate the liagase, since it is beeived that some ligase still sticking to the recombinant plsmid could reduce its transformation efficiency(i am not sure about this either)
4) or first subclone your pcr product in zero-blunt topo vectors, if your primers do not have 5'-phosphorylation and then cut the plasmid with desired restriction enzyme (just two days extra easy work/time) and then clone it again to your fianl vector. The idea is your restrction enzyme could cut your insert already in that topo-plasmid, better than a PCR product at its terminus, although it can bear extra overhang. if u have topo-TA kit, then do a pcr taq polymerse (dont use proof-reading DNA polymerse), to aid 3'A addition at ur pcr product to facilitate sub cloning by top-TA method and then repeat as said just above.
hope something fits as a solution to your problem. gud luck
is it possible that your self aren't competent enough. Are you using home made cells? In situations such as this I would recommend that you use company made ultracompetent cells such as Oneshot Genehog from stratagen. It cost an arm and a leg but they are very good. Ligation work that exceed 10kb start to become a little difficult.
At the level of the ligation mix, I would suggest that you use more concentrated DNA. (More vector and insert in the same volume.) That might help a little.
And before I forget, does the DNA ligate? As shown by formation of high molecular weight banding, when the ligated DNA is run on a gel. You should look at that just in case the ligase enzyme had gone bad.
Is there anyone who have experience in ligation of the large insert into a large vector? Thank you.
hi,
Mainly in such situations, the problem comes from the pcr insert digestion. i dont know what kind of controls you keep to ensure that ur pcr terminus is efficiently cut...
1) if your primers bear restriction site, ensure if they also provide sufficient extra overhangs that can support effective restriction digestion at the pcr product terminus. Ensure optimal digestion conditions for the restriction enzymes used to cut the pcr insert. you can increase the enzyme concentration and/or incubation time (with permissible final glycerol concentration to prevent any star activity ). check optimal buffer for double digestion conditions or try sequential digestion to ensure both the termnus is cut efficiently. For optimal overhangs for selected enzymes, go through
http://www.embl.de/ExternalInfo/protein_un.../extensions.pdf
2) you could also try reduced vector concentration, and/or final vector/insert concentration that helps in circularization of your 11kb vector to meet with the insert at its ends. 3) increase ligation incubation time 4) heat inactivate the liagase, since it is beeived that some ligase still sticking to the recombinant plsmid could reduce its transformation efficiency(i am not sure about this either)
4) or first subclone your pcr product in zero-blunt topo vectors, if your primers do not have 5'-phosphorylation and then cut the plasmid with desired restriction enzyme (just two days extra easy work/time) and then clone it again to your fianl vector. The idea is your restrction enzyme could cut your insert already in that topo-plasmid, better than a PCR product at its terminus, although it can bear extra overhang. if u have topo-TA kit, then do a pcr taq polymerse (dont use proof-reading DNA polymerse), to aid 3'A addition at ur pcr product to facilitate sub cloning by top-TA method and then repeat as said just above.
hope something fits as a solution to your problem. gud luck
Hi, Nagu,
Thank you for your reply. I agree with you that the problem comes from PCR insert. Last night I worked until midnight, I found there are 20% positive bacterial colonies in my current transformation( using PCR assay). Today I extracted plamids and am doing the digestion. I think maybe I finally get the assembled vector I want. But I have a question: why PCR products especially the large fragment are more sensitive to the the damage of UV exposure than others like plasmid?
Thanks again!
Wuyu
Wuyu
I am not sure if the linear PCR product and the closed circular plasmid ,have different frequencies in allowing Thymine-thymine dimerization. Such T-T dimers affect transformation efficiency and may be could even mutagenize or poorly express the selective marker, lacking any selection to form colonies. Can you double check the literature, if the confirmation show different senitivity to UV, which is somthing new to me, as I always knew that basically any conformer of DNA is vulnerable to UV exposure.
The restriction digestion result shows all the PCR assay positive plasmids are right one I want. So, I feel at ease.
I will check the literatures about the UV damage. Thank you very much. Next, I will do point mutation in this vector. Can you give me some advice?
Hi Thanks. I have not done site directed mutagenesis on bench.Better you can read about Gene tailor-product
http://www.invitrogen.com/content.cfm?page...469..Stratagene, promega also have some kits on that I guess, pls check it out..