I am cloning a beta-lactamase gene in pET28a vector. I have done PCR with deep vent polymerase. My next step is digestion followed by ligation in pET28a vector.
What is better? PCR clean up or gel extraction of my PCR product? My PCR product shows a single sharp band. Gel extraction process will cause more DNA loss than PCR clean up.
Your suggestions.
If you are 100% sure about your PCR product clean band without any sort of smear, in that case you can go ahead with PCR clean up. However if you you have any doubt, you would rather do gel extraction.
One thing you can do is, you already have gel cube with PCR product in it. Why not extract DNA out of it and see how much you loose and also purity. In this way you can compare results from two scenario and optimizemethod for down stream application.
Gel extraction is preferable, but avoid long exposition to UV.
I prefer to do a clean-up -preceded perhaps by a Dpn I digestion to eliminate traces of dam-methylated template, if applicable- and skip gel extraction altogether. If your target amplicon is the major band (especially in your case, where the amplicon has to be digested anyway prior to cloning), most of the recombinants you isolate will bear the 'correct' band. Gel extraction is not worth the hassle, if you ask me.
Thanks to you all for the valuable information. I have proceeded with PCR clean up for the amplicon (since I had single sharp band ). I have planned to use gel extraction protocol after double digestion of my vector and insert (amplicon) to avoid frequent exposure to UV rays.
Thank you Kunjan Desai for your suggestion to check the efficiency of extraction. We have observed that there is always a loss of approx 20% DNA during each gel extraction process. So, if the concentration is not too good, gel extraction would be a big concern.
You can use PCR purification method after double digestion too! In principle, the column that binds DNA in both method is the same! so fragments smaller than about 20bp will be washed off!