Dear Friends, I had tried everything for my ligation.
4degree overnight
8degree overnight
16degree overnight
During PCR also, I had kept extension time up to 20 min. My PCR gave crisp bright band and after gel extraction also nanodrop also gave good concentration. but after transformation blue-white colony are coming but when I am doing colony PCR with M13 primers And Gene-specific primers. It is showing no insert. I have checked at every step.
According to me, after gel extraction, I am losing PolyA-tail. So I want to ask Is after gel extraction, shall I keep for rePolyA tail extraction?
Personally I"ve never heard of A lost in DNA fragments but I can't be sure if its impossible. Are you using a DNA polymerase that adds A to DNA extremity? Taq and Elongase do add but enzymes like Pfu and Phusion don't.
There is a simple protocol for A additon to DNA fragments using Taq. You just have to perform the 72oC extension step for 10min in the presence of dATP (you don't need primer).
If you are getting a lot of colonies, but none have insert, there could also be a problem with your recipient vector. Perhaps the T overhangs have been lost. You might want to get a new batch. I would also consider doing a few minipreps and restriction digests to be really sure there is no insert. Competition with genomic DNA might impair PCR that works in other situations. Finally, you can try to do the ligation with the PCR product without purifying it first as long as the PCR template wasn't a plasmid or was a plasmid with different antibiotic resistance than the vector. Side note - Taq adds a single A to the end of each strand. PolyA means many A's in a row and a polyA tail usually refers to the modificiation added to the ends of mRNAs.
Michael thanks a lot for your reply
Sir, I am sorry I meant to say A overhang. Sir, I tried with new batch of ptz-vector and pGEMT vector but still same story. Sir, I also have one doubt, if T overhang from vector had gone then how blue and white colonies were coming? and suppose self-ligation of vector also occurred then also In M13 PCR something should come. No band is coming in Gene-Specific and With M13 also.
The theory is that some vector plasmids lose the T overhangs which allows a blunt self ligation, giving blue colonies. I haven't worked with pGEMT, but I took a quick look at the sequence. I believe the M13 PCR without insert should produce about a 200 base pair band. PCR from white colonies should produce a product the length or your insert plus 200 base pairs. If you don't see either PCR product from blue or white colonies, the colony PCR may not be working. I would definitely try a restriction digest. There are EcoRI sites on both sides of the cloning site you might use. Another question is whether you do a control ligation without insert DNA. If this produces a lot fewer colonies than a ligation with insert, it would also indicate that the ligation really is working. Good luck!
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