facing problem in cloning.No colony in transformants plate.
How to check ligation is worked or not.
Choice of plasmid is correct.
double check also your plate and prepare fresh plate every time for the cloning transformation.
After the transformation, which media do you use for the incubation??
Riccardo
@riccardo..there is no colony in plate.i.e no transformation..
Media used is LB
I was talking about the media that you used after the thermal shock/ elettroporation. So in theory you transform your cell then you incubate at 37C for 1 h then you plate. During the cloning when i used the LB for the incubation i didn't have colonies. Try to use the SOC media or other more nutrient media. you can also increase the 37C incubation time before plate your cloning
@Hua Xao...what do you mean by before ligated sample.plz explain
Did you perform a digestion before the ligation ? If so, did you checked this digestion worked properly ?
Is that a double digestion of your plasmid and your insert ?
In that case, I usually perform 1 experiment and 2 controls for each DNA : the same conditions with no RE, 1 RE and the 2 RE. In fact, I had several troubleshooting which came from enzymes that didn't work properly, probably because it was and old stock.
Also, I prefer to separate digested DNA on a gel and then performing a gel extraction in order to get rid of contaminations (non digested or partially digested DNA)
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning