PCR product purification
|
PCR product purification (archive)
After PCR with primers containing restriction sites added on, can you
immediately digest the PCR fragments prior to purifying by gel
extraction? In other words can you avoid an extre gel purification or
PCR clean up step prior to restriction digest of the fragments? ... ...
Last update 06-Aug-2007, Rating Good of 2 votes.
|
Write your comment
|
je voudrais avoir un protocol de purification des produits PCR à partir du gel d'agarose. il s'agit de fragment de taille de 650 à 1100pb
Rating: n/a
Reply
|
|
|
Hi there,
If you're considering using your PCR product as starting material in a restriction digest, you just need to make sure that the final buffer composition of the digest is appropriate for the restriction enzymes you're using. If you're using a large volumem of PCR product in your digest, check the composition of the PCR buffer. Do you need to add less Tris or other salts to make up for the salt already present?
The gold standard is to try the digest without purifying your PCR product, and compare that insert's ability to form colonies after ligation with a purified insert's ability to form colonies. If there's not much of a difference and you get positive colonies even when you skip the purification, then your purification wasn't doing much to begin with.
Rating: Excellent!
Reply
|
|
Related resource
PCR purification and storage
PCR products electrophoresis
|
