Low Transformation Efficiency with a Specific Gene - (Jul/15/2013 )

Hello everyone.

I cloned the gene Syncollin (400 bp) into a vector and transformed it into E.coli. But the transformation efficiency was very low. I picked up 6 colonies from the plate and inoculated them. Only 1 colony had the insert, and unfortunately with some mutations. I repeated the cloning procedure from scratch, the result was similar. The thing is I got really good transformation efficiencies with other genes with the same vector (e.g. amylase - 1.5 kb, GFP - 700 bp, etc). Also for the ligation step, I always use 3:1 insert:vector molar ratio. So what do you think the problem might be caused by? Have you ever experienced such problem with the cloning of a specific gene?

Thanks.

You can also consider the problem as low ligation efficiency.

It could be any one of a number of problems that can occur during cloning and transformation.

What can you tell us about this gene (is it G/C rich?, secondary structure?).

How are you generating the insert (if PCR, which polymerase)?

Which restrictions are you doing? For how long? How are you cleaning them up?

Are you dephosphorylating the vector?

Could the gene product be toxic to ecoli?

How did you confirm the presence of the insert?

Did you try any other ratios?

etc...

Given your success with other genes, it seems the most likely situation is that the gene is toxic to E. coli. This would explain why you are seeing versions with mutations, which would presumably reduce or eliminate this toxicity.

Hi Mevcit,
Beside the professional suggestions of Bob1 and Phage434, I would like to share some of my experience:
1. If the transformation efficiency you mentioned was the very few colonies you got, it was likely your insert is toxic for cells.
2. If the efficiency was that you got lots of colony without insert, it was likely because your ligation step had problem.
For the second reason, I would like to suggest you to:
- Increase the restriction time (4 hr or overnight) or add more RE into the reaction tube. Your vector might be not cut completely
- Increase the ligation time (I usually used overnight at 16oC, it works better than 2 hr at RT, I can got 100% colonies with insert). However, the condition depends on the manual provided with the enzymes you use.
Good luck!