What could be the most probable reason for not getting colonies on Amp plate?
Many reasons are there, either from PCR reaction or plasmid is not suitable to the organism you need to clone, I advise you to read more literature related to your work.
Regards
On the top of the previous answer, these are other possibilities:
Incomplete digestion, failed ligation, old compenetnt cells or not sufficient transformation.
I included controls for linearization and ligation but how for transformation?? James.. plz elaborate
Danijela probably ligation is failed or transformation is insufficient? what should i do next ? How to sort out this prob?
I am assuming it is blunt-end ligation or did you digest the PCR Product?
If it is a blunt-end ligation, what Polymerase did you use for the PCR product? Taq based Polymerases will give you a 3' adenine overhang. This will interfere with your ligation later.
Did you dephosphorylate the pfbdm plasmid? If so, is the PCR Product phosphorylated?
To check whether the ligation is perfect or not, you just take about 50 ng of ligated product and run with the linearized plasmid DNA. You can clearly see the shifting of the ligated product. Then transform the DNA. Another thing is, you check the transformation efficiency of the competent cell.Always try to use fresh competent cell for DNA transformation.Another point is that, change the vector:insert ratio.
Thanx Sebastian, James, and Puspita .. I am doing USER cloning followed by transformation in Top 10 cells. PCR product is not digested.. i am trying with different insert:vector ratios.. however still not getting coclonies..
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning