RE Double Digest XhoI/NdeI TA Vector - (Jan/24/2006 )

Hi guys,

I'm attempting to cut my DNA insert (~627 bp's long including RE site) out of a TA vector using XhoI and NdeI. My previous two attempts have failed. Below are the outlined protocols I followed:

Attempt 1:
-5 uL react 2 buffer (invitrogen)
-3 ug DNA (ta vector + insert)
-y H2O (to bring total rxn volume to 50 uL)
-0.5 uL XhoI (invitrogen)
-1 uL NdeI (invitrogen)

-mix buffer, dna, and h2O before adding enzymes
-2 hr incubation time @ 37 C

Attempt 2:
-5 uL buffer 4 (NE biolabs)
-5 uL BSA (NE biolabs)
-1 ug DNA (ta vector + insert)
-y H20 (to bring total rxn volume to 50 uL)
-0.5 uL XhoI (invitrogen)
-1 uL NdeI (invitrogen)

-mix buffer, dna, and h2O before adding enzymes
-1.25 hr incubation time @ 37 C

Per NE biolabs technical website advice and previous attempts by another researcher in our lab, attempt 2 should of and has worked in the past. However, I've not been as fortunate. Let me mention also that I PCR amplified the insert out of the TA vector to check for its existence before proceeding with the double digest.

Any pointers? Bad enzymes? Longer incubation time?

Any advice appreciated!

Thanks in advance,
WhiskeySour

BSA is 100X
so adding 5µl would give a 10x final solution

XhoI

*sigh*

This is becoming an obsession with me.