I have a clone of my gene in PGEMT. I did restriction digestion and tried to elute it. Similarly I did restriction digestion of my expression vector to create sticky ends. The concentrations of elute are very less more over the 260/280 ratios are altered. I did ligation with t4 ligase and after transformation in competent cells I did not get any colonies.
Any solutions?
Even if you get less DNA concentration after gel purification. Try to speed vap it to reduce the volume. Set the reaction in lowest volume possible like 10 ul at 16 degree overnight and then transform the whole soup.
to get the clone in first attempt, you should have high concentration of your gene of interest as well as your expression vector. quantify your DNA samples first and when you get the good amount of DNA, you can calculate the ligation reaction volume by ligation reaction calculator (free available). set up the reaction accordingly and transform that reaction in pre-checked high quality competent cell(competency of the cells play major role in transformation ).
Best of luck
Competent cell efficency is good. I have used the same competent cells to clone my gene in PGEMT. The problem I am facing is the elution of my vector and gene after digestion from gel. The concentration is too low like my gene has concentration of 17ng/uL and vector 11ng/uL.
Again I calculated the insert required per ligation reaction with the formula
(Ng of vector×bp of gene/ vector size ) ×3/1 and similarly for other ratios.
I did ligation for an hour with t4 ligase and then heat inactivation followed by transformation in competent cells.
The observation is that my concentration of elute and vector is too low and the ratios 260/280 are altered ranging from 3 to 5. Is there a problem if I use this for my cloning experiments?
Please help
@Priyanka can you please suggest something after reading my above comment?
We have worked with such low concentrations and it worked fine just that while setting up the ligation mix in 1:5 ratio keep reaction volume as low as possible (10ul) and keep it overnight at 16 degree. It should work. After all that, if still don't see any transformants try changing your t4 ligase or buffer.
For calculation of ligation mix you can use neb calculator.
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