I´m new here und happy to have found this forum.
In future, I will also try to help other users with their questions.
I hope you can help me with my ligation problem.
I´m trying to ligate a 1 kbp Insert, which was cut with EagI und SpeI out of the TOPO-Vektor (pCR 2.1 from Invitrogen) into a vector about 9 kbp, which was also cut with EagI and SpeI.
I did restriction 2 hours at 37°C, then added CIP to the vector and incubated for one more hour. Then I gele purified my desired products (Qiagen Kit) and did the ligation with T4 DNA Ligase.
I´ve tried over night ligation and for 3 hours at 22°C (it´s a sticky end ligation).
For the transformation of competent DH5alpha I´ve used the whole 20 µl ligation approach (heat shock for 30 sec at 42°C).
So the problem is, in over 20 ligation and transformation trials I don´t get a single colony.
The strange thing is, I did a negative control (only vector with ligase without insert) and got a bunch of colonies!
I´m asking me, why I don´t get any colonie, at least I would expect colonies with
1) closed vector (for the case CIP didn´t work at 100 %) or
2) open vector (for the case the ligation didn´t work properly)
Why do I get colonies with vector only in my negative control but not in my ligation approach?
Things I´ve already changed:
-new T4 DNA Ligase
-new competent cells
-new LB plates with ampicillin
-vector:Insert ratio vom 1:1 to 1:5
Thanks for your help!
Edited by Nose, 03 May 2009 - 06:18 AM.