I have gotten great results by purifying my PCR product with a Qiaquick PCR cleanup column prior to cloning. Just make sure you run a bit on a gel to know if you have the right product size (and only one band).
Yes I do! The gel purifying step allows the elimination of any unwanted bit of DNA generated during digestion of vector and insert which may interfere at the ligation step.
About PCR digest : at least 3 hours respecting the recipe provided by the enzyme manufacturer as there is no easy way to check the digest efficiency...
I have gotten great results by purifying my PCR product with a Qiaquick PCR cleanup column ( Qiagen) prior to cloning. Just make sure you run a bit on a gel to know if you have the right product size (and only one band).
Concerning PCR digest : at least overnight respecting the recipe provided by the enzyme manufacturer as there is no easy way to check the digest efficiency...
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I advise you to use the TA cloning technology ( insert your PCR fragment in an intermediary plasmid.
I use gel-extraction method. It works well for me. For digestion, around 3 hours for single RE (both ends with the same restriction sites) digestion or double digestion with a common restriction buffer.
After the digestion you can purify your PCR product using phenol-cloroform sequence. You start with the same volume phenol, then half and half phenol-chloroform and finally the same volume of chloroform. Always collect the upper phase. The phases are separated at 15.000 for 5 min at rt. Precipitate your product for 1 hour in -80 by adding 2.5 volume of ethanol and 1/10 to 1/20 CH3COONa ph 5.2.
Gel extraction is fine when non-specific bands are there. If amplification produces single band of one's own gene of interest, one should go for PCR purification.