4 replies to this topic

[johnk]

Hi all,
I have been using TA cloning kits for couple of years but due to the high cost I wanted to switch to blunt end cloning.

I took religated pGEM T Easy vector, cloned it, extracted the plasmid and digested it using EcoRI to get blunt end vector. I dephosphorylated the vector.

I use Taq polymerase for PCR so I incubated my PCR product with Klenow to generate blunt ends. I followed the ligation as usual but I got no colonies.

What could I be doing wrong?

[phage434]

For starters, EcoRI does not produce blunt ends.

You can make your own TA cloning vector, which might be a better approach (my opinion).

[tfitzwater]

1. You should have used EcoR V rather than EcoR I.
2. If you use CIP or BAP to dephosphorylate, you must be extremely accurate in determining the concentations of DNA ends and use exactly the recommended amount of enzyme (this varies by supplier).  SAP is much safer and can be used in the restriction digest.

[johnk]

Sorry I made a typo. I did use EcoRV to get blunt ends and I used SAP to dephosphorylate it. Is there a protocol to create your own TA cloning vector?

Edited by johnk, 22 June 2010 - 08:44 AM.

[Curtis]

I use CloneJet from Fermentas for Blunt end cloning. It has a reagent to blunt end your PCR product in case it has overhangs. It does it in 5 minutes, and also the ligation is about 10min to 1 hour. It is very quick and efficient.