3 ways ligation for cloning
Posted 14 September 2010 - 03:52 PM
My setup is
1] Vector pEt 15b digested with Nde1/Xho1 and dephosphorylated and cleaned up with qiagen kit
2] Insert 1, 210 bp from pcr product digested with Nde1/Bamh1 and cleaned up with qiagen kit
3] Insert 2, 210 bp from pcr product digested with BglII/Xho1 and cleaned up with qiagen kit too
I have tried a molar ratio vector/insert 1/3 without success
Have you some advices for me
Posted 14 September 2010 - 06:44 PM
If you must use these enzymes, I suggest you TA clone your PCR products first, then liberate them from the TA vector by digestion, gel-purify the fragments, and then try them in your 3-way ligation.
Posted 15 September 2010 - 07:38 AM
Have you some advices for the insert/vector ratio i will try 1/! and 1/10.