2 replies to this topic

[anonymous]

From: Vertino P.
email:
Category: General method

Message

Dear netter,

I have amplified my pcr products with primers which have a restriction site introducedinside 4 extra nucleotides, and have tried many times to clone my pcr products (afterdigesting with enzymes) into a plasmid cut with the same enzymes. it simply did not work.Can anyone give me a clue?

Thanks in advance.

Vertino

[anonymous]

From: Samir
email:
Category: General method

Message

I think the posible cause may be the enzyme you used to cut your pcr products doesn'twork well with 4 extra bases outside it's cutting site. you may have to add 2 or 3 morebases to your primer or TA clone your pcr first then subclone into your expression vectoror any other vector.

Good luck

Samir

[anonymous]

From: craig
email: craig.mcandrew@kcl.ac.uk
Category: DNA-Related

Message

It's true that it is possible that your enzyme does not cut efficiently with a fourbase overhang, you can check in the back of the N.E.B. catalogue. It may also beworthwhile to digest your PCR products with a vast excess of enzyme, the units of activityrefer to ug's of lambda DNA with is something like 20kb.