Hi,
I am cloning a 2.8 kb gene into pet28b vector. I did two ligation reaction. In one of them, I did not put insert (only vector and ligase) (control reaction). Then I transformed 10 microlitre of both onto Dh5a. I got 4 colonies in ligation plate while no colonies was present in control plate. I inoculated 4 colonies from ligation plate in LB (with kanamycin). I did plasmid extraction from kit but in all 4 of them I got very low yield (something around 2.8ng/ul). I am in doubt whether plasmid is there or not?? Why is this happening???
Hello,
You should consider doing a colony PCR ideally with a region of your vector and your gene to verify the plasmid was successfully transformed and was carrying the gene. (You could also run it on the yield directly or do a restriction enzyme digestion to verify if is correct). If it is correct, your low yield may be due to a problem with your extraction kit/technique.
I agree with Kevin, you should do a colony PCR to confirm if your insert has in fact been ligated into the vector.
Also, you might be adding too much of the ligation mixture into the competent cells. The ideal volume to use is 1-5µl or the transformation efficiency of the cells is reduced.
Shruti Jain
www.inspiralis.com
Hi Safdari,
Is your objective protein expression (because I see that you used pET system)? In that case, do not worry about the yield. Just confirm whether the colonies are positive and retransform the positive constructs into BL-21 or rosetta cells (of your choice). If you wanted to increase yield, consider midiprep. There are several reasons for low yield: insert toxicity, addition of low or high amount of Kan, etc. I can suggest more based on your additional details.
Hi,
I doubt that the 4 colonies you got have the desired insert. If comp. cells are good, your transformation should end with 200 to 400 colonies with 50ng of vector ligation mix. I suggest you to focus on ligation mixture rather. Check the transformation efficiency of cells with empty vector (1ng).
Regards
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