3 replies to this topic

[Nose]

Hello together,

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 enzymes
-new T4 DNA Ligase
-new buffers
-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.

[Danina]

Hey Nose,

Which T4 DNA ligase did you use? in my lab, we have Promega T4 DNA ligase which ligate best overnight at 16 degree. See if 22 degree is the right temp for your ligation reaction.

If you have no colonies, one of the possibilities is that your bacteria didn't have the Amp Resistance gene which would have come from your vector if the bacteria is taking up the vector, and hence the Amp on your plate will kill them.

Did you incubate your DH5alpha and ligation products on wet ice for 30 mins before heat shocking it?
Try to heat shock your DH5 alpha and ligation products for 90 secs instead of 30 sec and then put it back on wet ice for 2 mins. After that, add some LB broth to help the bacteria to recover from the heat shock (1 in 10 dilution). Then shake the solution in a 37 degree incubator (I usually use 220 rpm as the speed) for one hour before plating your transformation reaction overnight at 37 degree.

Hope this helps
Dani

[Nose]

Hi Danina,

i have used the ligase from Promega and Fermentas.
The ligation vector has amp resistance and I know that the reason of getting no colonies is due to the non-uptake of the vector.
I´m wondering why the bacteria don´t take up at least the vector without insert as in my negative control.

What you suggest is the standard protocol for heat shock and sure, I did it that way. I have also changed the heat shock time from 30 to 90 seconds without result!

[Danina]

Hi Nose,

the bacteria won't take up your vectors because it's in a linear form, not circular. For my ligation, I only use about 50 ng worth of vector, hence my negative control turns up empty. If you start up with something like 1 mg of vector, then yes, you will have some colony growth on your negative control plate. What I am saying is the bacteria will take up the linearised vector but the efficiency is low, hence why you don't have any colonies on your negative control.

Did you check whether your digestion has work by gel electrophoresis?

Have you consider changing the vector to another type?

Dani