Blunt end ligation
Posted 21 June 2010 - 06:12 PM
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?
Posted 21 June 2010 - 07:23 PM
You can make your own TA cloning vector, which might be a better approach (my opinion).
Posted 22 June 2010 - 06:50 AM
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.
Posted 22 June 2010 - 08:43 AM
Edited by johnk, 22 June 2010 - 08:44 AM.
Posted 23 June 2010 - 08:36 PM