3 ways ligation for cloning - 2x 210bp fragments and pET 15-b (Sep/14/2010 )
I have troubles to make a 3 ways DNA ligation
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
Neither NdeI nor XhoI cut very well near the ends of PCR products (see here). You problems are probably due to these enzymes not cutting your PCR product efficiently, so you don't have the right ends to clone them into your vector. Did you engineer these sites into your PCR primers? How many irrelevant 5' bases did you include in your primers upstream of the restriction sites?
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.
Ok thanks for your fast answer, I will try soon the pcr blunt vector which we have in my lab, because to obtain my pcr product I have used pFu polymerase.
Have you some advices for the insert/vector ratio i will try 1/! and 1/10.