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Subcloing PCR fragments - (Nov/27/2005 )

I am new to moleq bio and have some questions.

I have PCR'd my fragment of interest, looks good on a gel.

I will double digest the product leaving 1 sticky and 1 blunt end.

I'll do the same for the vector that I will clone the fragment into.

How should I treat the vector before ligation? - Dephosphoralate? (sorry about spelling)

After a transformation, and I streak on Lb/AMp plate how do I know what colonies have the insert. I could do PCR to confirm, but my goal is to make RNA?????

Very confused on thouhgt process.....


try this:

it is a product plug, but there is some useful info

i would do a screen of a number of colonies for the proper insert; you can use PCR of boiled cultures for a quick check, then go back and mini-prep a couple likely candidates and verify further. You can re-cut your product with the enzymes used in cloning to verify the presence of your insert, and you can also sequence the clone (best bet to be sure)

there are some protocols on protocol-online that cover these topics; you can also search the web for cloning tips for more details

good luck