i want to cloned the promoter sequence of about 2kb length, i did several attempts to amplify that genes sequence but failed after each, kindly suggest me the way out to get successful amplification, or i can send the genes name or sequences for critical evaluation
It is very difficult to diagnose a failed reaction because it could have failed at any one of a number of steps. Tell us what controls you did and that might help, or else do some appropriate controls. The things to consider are whether your PCR reaction mix and polymerase are still good, are you sure your primers are correct, do you have the right conditions for the amplification, is your template DNA good. The more information you provide about the experimental design and what you tried then the higher likelihood that someone can give you specific answers.
Primer dimer? Tm? Polymerase used? High GC? Any/all of these can affect success. Hard to say what the issue is when we have no details.
Hi.
What are you going to do after amplifying that region? Are you use it for cloning? What is the size? I am asking you this because ordering a company to synthesize a sequence for you is not expensive. Even you can optimize the sequence, add some extra tails for subclonning and around 1000 bp is only around 200 dollars, so just keep that in mind
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning