To avoid an additional cloning step, I want to use whole plasmid PCR.
I have designed my primers back to back (i.e. non-overlapping) and they are located in the origin of replication of my plasmid. I have checked the PCR product on a gel and it shows the correct product (linearized and the full plasmid length) but it also shows some other non-specific bands. I thought I don't need to be concerned about these bands since once I recircularize/ligate, only full plasmids with an intact origin of replication should be viable.
I have done this recircualrization in the past where I have removed all the nonspecific bands and kept the purified PCR product at a low concentration and the T4 DNA ligase at a low concentration and had it work fine (i.e. I get good transformation efficiency into E. coli).
This time, however, after transformation via electroporation, I am not getting very many colonies, so I think the recircularization did not work. The product had been cleaned using a PCR cleanup kit, but the nonspecific bands were large enough to be retained.
Could the problem be caused by the presence of nonspecific products in the ligation reaction? Is there a better-optimized way to recircularize if the product has blunt ends?
Thank you.
Hello,
In general smaller DNA molecule ligate faster than the larger molecule. If the nonspecific bands observed in the PCR are smaller, than it is hard to get the expected ligation products. This can ge avoided by simple gel elution method.
Good luck
Have you phosphorylated your PCR product or used phosphorylated primers? Otherwise you don't have substrate for the ligation reaction.
Recicularization is indded very efficient, but as Michael already pointed out: There is no ligation without at least one 5'-phosphate.
Do I miss something-I do not get shat you want to achieve by your PCR.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques