I am trying to clone an E.coli gene of 2106 bp into an expression vector.
The first problem faced while cloning is that the PCR is not getting optimised. 3 Non-specific bands are getting amplified along with the desired product; even when the band of interest is gel-eluted and then amplified. Second problem is that, the PCR product which is gel-eluted, after restriction digestion is giving rise to two bands. one above 2Kb and another just 100-200 bp less than 2Kb. The primers bear the restriction sites for BamHI and XhoI. I have checked in NEB cutter website that the gene does not contain the restriction sites for BamHI and XhoI. Can anyone please help me what is going wrong?
It can be possible that while cutting out the region of interest accidentally you might have picked some non specific region. For your second problem try running the gel at a very low power like 15 amp/hr for a very long time like 7-8 hr (overnight). This will help very good separation of the two bands. Once resolved well, cut out the desired band and go ahead with your cloning. Forget about the rest of the PCR problems.
If you PCR product is giving 2 bands following digestion, and the gene does not contain these bands, you are most likely extracting a non-specific product. If possible, could you send the insert for sequencing?
Secondly, to optimise PCR, try to perform a temperature gradient, a MgCl2 gradient, and even a pH gradient. If those don't help, but the band that you're amplifying is the most specific one, then I would suggest that you give touchdown-PCR a go. This uses a high annealing temperature to begin with, and with every subsequent PCR cycle, the annealing temperature is decreased by 1 to 0.5 degrees, allowing for the most specific products to amplify during the initial cycles of the PCR. This then outcompetes any non-specific products, and can result in a much cleaner PCR.
http://bitesizebio.com/2203/touchdown-pcr-a-primer-and-some-tips/
Juliette Delhove Thank You. But is there any specific PCR machine requirement for performing touch down PCR? We are using Eppendorf Mastercycler.
Hi Sagarika,
No, there is no specific machine. I also use the Eppendorf Mastercycler. You can add a 3-step cycle, and then where you usually put a gradient, uncheck this box, and instead press the +/- button at the bottom of the machine so that it reads decrease by 0.05 degree C or 1 degree C. Then add from which temperature you would like to start and until what temperature you would like it to "touchdown" to. I usually do mine from around 65 to 55 degrees. Then you add another 3-step cycle and add your regular amplification parameters using the annealing temperature that you think will work. Then add your final extension, and that should be it. Best of luck and let me know if something didn't quite make sense. It's a bit tricky to explain the PCR machine parameters in writing, but just take a look.
- Badges
- Science method