How long do you digest your PCR?
Do you believe cloning efficiency improves when isolating the band on a gel, rather then simply purifying it?
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).
Thank you, so you simply purify after amplification and before digestion.
After digestion do you heat inactivate (if feasible) vs. Purification?
Hi there,
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...
or
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.
From my experience the following always worked:
* Do a PCR, 4x50 µl to get plenty of DNA
* Purify on a single column
* Digest for 1.5 h (NEB CutSmart Enzymes)
* Run on an agarose gel, purify again
* Ligate (Fermentas RapidLigation Kit) for 1h at 22°C
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.
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