Hi; I'm trying to optimise my protein expression. I have lighted my product into pET32b vector and transform into TOP 10. My objective currently to subclone them into bl21 De3 for protein expression. However so I am trying to confirm the insert via PCR. I have the desired product but the intensity is very low. Hence hardly can see a product in digest. Please help how to over come this matter. Attached is the gel photo. The red marks the appropriate band.
Can you confirm the steps you took leading to that gel? Skip over the transformation part. How long did you leave the transformed cells to grow? Did you do a colony PCR or did you extract the plasmid from the cells, then PCR? If the later of the two how much plasmid DNA did you get back?
What are your PCR conditions? Did you isolate the plasmid prior to PCR, or did you run a colony PCR? There is a potential that you just need to increase the number of cycles for your PCR.
You have a very strong band of primer dimer. Please lower the concentration of primer or increase the amount of template for PCR.
Or increasing the annealing temperature
I agree with Alex. You seem to have a lot of primer dimers in your reaction. You might want to double check the final primer concentration you are using in your PCR reactions. I routinely and successfully use 500 nM of primers and 50 ng of DNA template in 50 ul reactions. Since you do not seem to have a good amplification of your desired insert, you might want to try small-scale (20 ul) gradient PCR reactions first to find the best annealing temperature for your primers and then scale up the reaction accordingly.
- Badges
- Science topic
- Similar topics
- Computer Science and Engineering
- Bioinformatics
- Bioinformatic Tools