Ligation into pET28 - (Jan/26/2007 )
Hi,
I try to ligate an Insert (800bp) into a pET28 vector (6'000bp). The insert was amplified with PCR and the restriction sites added with primers (next to the restriction site there was an overhang of at least 5 bp). PCR and digest reactin was purified with PCR Clean Up Kit from Sigma (eluted with water to not have toxic buffers). Ligation was over night at 16°C. After transformation into chemocompetent cells I never get more than 4 colonies per plate (with the appropriate antibiotic of course). I also tried quick an dirty ligation on the bench an different ration, but I never got more than only a few colonies (and all negative 
).
So, what can I do?
Thx
Have you checked the concentration of your eluted DNA and have you checked the pH of the water you elute with?
Jep, concentration is always checked (NanoDrop or spectrometer).
I use destillated and sterile water. pH should be normal. I never had problems with it
All ready, you have differents points to chek:
- strategy of the cloning ( vector and insert sequences)
- transformation with chemocompetent cell (cells or protocol!)
Try again but with control + and - of ligation and transformation.
Also check if you are not overdigesting the vector and PCR product. As overdigestion could be problem with some enzymes.
Are you cleaning up the PCR reaction up prior to digestion? If not, then that is the problem, since the PCR enzymes, buffers, and dNTPs are all waiting to extend the 3' end of your freshly cut DNA, making it impossible to clone.
Also, water, no matter how pure or sterile, is not a good thing to elute DNA from columns with, since it has very poorly controlled pH. Use 10 mM tris HCl pH 7.5 or 8.0
Totally agree. Something a lot of people don't know is that mQH2O is actually around pH = 5.0 believe it or not. Not sure why exactly.
Totally agree. Something a lot of people don't know is that mQH2O is actually around pH = 5.0 believe it or not. Not sure why exactly.
Answered by phage434
http://www.chem.usu.edu/~sbialkow/Classes/...nic%20Acid.html
In general, the pH of pure water is very poorly controlled. Micromolar or even nanomolar amounts of virtually any ionic compound will dramatically change its pH. There is a reason we use buffers in biochemistry.
Totally agree. Something a lot of people don't know is that mQH2O is actually around pH = 5.0 believe it or not. Not sure why exactly.
Answered by phage434
http://www.chem.usu.edu/~sbialkow/Classes/...nic%20Acid.html
In general, the pH of pure water is very poorly controlled. Micromolar or even nanomolar amounts of virtually any ionic compound will dramatically change its pH. There is a reason we use buffers in biochemistry.
Ha, yep, that was my post. I knew about bicarbonate but still had it in my head the pH was 7.0. Damn pH equations.
Thx @ all
I'll try.