Identify insert by restriction digestion - (Aug/07/2006 )
Hi,
I try to insert a fargment about 400bp to pET 15b from Novagen. I cut the insert and the vector with the same enzyme: Nde1. My question is how do I check the insert? How do I figure out which enzyme I should use after the mini-prep? Tks
I try to insert a fargment about 400bp to pET 15b from Novagen. I cut the insert and the vector with the same enzyme: Nde1. My question is how do I check the insert? How do I figure out which enzyme I should use after the mini-prep? Tks
You could digest with NdeI, or do a PCR using primers from the plasmid. Check the plasmid map, it should have some suggested primer sequences to do this very thing.
Digest the minipreps with NdeI should release the 400bp fragment. If there r other enzymes in the map beside the insert, should also work.
I try to insert a fargment about 400bp to pET 15b from Novagen. I cut the insert and the vector with the same enzyme: Nde1. My question is how do I check the insert? How do I figure out which enzyme I should use after the mini-prep? Tks
Thank for yr advice. But the problem is how do I know it is the right direction? Must it be the internal enzyme ?
best would b to sequence it. Or cut inside the fragment to give u some idea.
If you don’t know the sequence of your 400 pb fragments, you have to sequence. If you already know the sequence, look for an enzyme that cut near of an extreme (In your fragment) and with an enzyme that cut ones in your vector and do the double digestion 
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Thank for yr advice. But the problem is how do I know it is the right direction? Must it be the internal enzyme ?
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You could always just pick a few colonies, and try some small-scale expression expts. Running a gel will tell you which ones are the right way around, and you get some data on expresion conditions. You might even find that the reverse clones do something useful!