I have been trying to insert a gene of interest in pcr4TOPO vector. Vector insert joining and the transformation was carried out as per the guidelines in the protocol ( Invitrogen ). Colonies which were resistant to ampicillin (transformants) was selected. PCR reaction was carried out to confirm the presence of the insert in the vector using insert-specific primers. But the appropriate band was not found in the agarose gel. That meant the absence of insert in the vector. And also a single band was obtained when the plasmid (which was isolated from the transformants) was run in the agarose gel. Does it indicate the linearized form of the vector and also can I use the isolated plasmid again for transformation assuming that the T overhang is still there?
Note: Instead of using a fresh PCR product for TA cloning, I used one month old PCR product which was amplified using Taq polymerase.
hi Srimeenakshi,
There could be many reasons for this.
1) Since you didn't get the PCR for insert, maybe the insert didn't get ligated with the vector and instead the vector self ligated and gave colonies on amp selection.
2) Since the insert was very old so may be it was already degraded before you started the cloning. did you check the amplicon on agarose gel before starting cloning.
3) check the plasmid with restriction digestion if your gene has some restriction sites at the flanking regions.
4) did you put any positive control for PCR when checking for positive clone. Maybe the PCR reaction has failed due to which you are not getting any amplification.
Hope this will help. if not then you may try to repeating the cloning.
yes sir, I used a positive control to check the PCR reaction. And as the topo vector has T overhangs at both 5' end, I ruled out the possibility of self-ligation also.
Hi Srimeenakshi,
did you try polyadenylation of your PCR-product? Of course in PCR products generated using Taq-Polymerase PolyAs should be present. But if you store the PCR product they might get lost. Just incubate the PCR product with Taq polymerase, its buffer and dATPs (final concentration of 0.2 mM) at 72°C for 20 min and try the cloning again.
Good luck!
thank you sir.. Alternatively, I can use freshly amplified PCR product as well right sir?.
Yes, of course! If possible do the cloning directly after PCR without freezing the product.
If I am you I would use a little more than what is required of PCR product in the ligation step and also of ligation mixture in the transformation step. Also would maintain a positive control. Plus heat shock the cells for +10 seconds over what is recommended. Good luck.
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