What are the possibilities of cloning a PCR product with standard Taq (TA overhang) into a PET vector (Champion PET 100 Directional TOPO ). The manual suggest that I make blunt end pcr products but I want to know if it will work with normal pcr product because topoisomerase should remove the overhang I suppose ?.
Dear Binoy, blunt end cloning does not need any overhang, and I am surprised why You are willing to use blunt end cloning method as that is very difficult and time-consuming. You are better to design primers with flanks of appropriate cut sites as those in your vector with enough distance from each other and absent in your insert PCR amplicon.
Binoy,
It means you need to not have a T/A overhang. The best way to do that is to use a a proofreading polymerase (PFU, Phusion,... ). Alternatively you can use a SS nuclease like the one from Mung Bean to chop the T/A off. Or you can fill in the ends using and "end repair" mixture, in which case you'll end up with a blunt product containing the extra T/A. Watch out what that would do to your reading frame.
Babak,
These topo vectors come predigested, pre-activated and are ultra-convenient. It's not at all like what you read in the books about classical "blunt-end cloning" (which uses T4 ligase, not topo).
Thank you guys for responding. May be I was not clear With my question. Let me quote from the Life Technologies site regarding the vector I am using, "Directional TOPO® cloning vectors contain a single-strand GTGG overhang on the 5´ end and a blunt end on the 3´ end. The four-nucleotide overhang invades the double-strand DNA of the PCR product and anneals to the CACC sequence that you place in your 5´ primer. Topoisomerase I then ligates the PCR product in the correct orientation" (please see attachment)
Now the issue here is I have a pcr Product With a A overhang at 3' end. My question will this overhang also be displaced by topoisomerase in the cloning procedure described above. I dont have a proof Reading polymerase at the moment so was just thinking if it should be fine With a normal taq.
Hi, CACC overhang is for directional cloning. When you amplify your target fragment, you need to add CACC to your primer. Then CACC will be complementary with GGTG of vector. so you can clone directionally. and your insert have to be Blunt ended. only blunt end insert can match with Blunt end vector. just like TA cloning. if your insert is A end, you have to use T end vector. if you use blunt end vector. you will get very few colony. one word insert have to match with vecor. T match A, blunt match blunt. or CACC match GATT. www.aidlab.com.cn
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