Hi!
I am trying to clone a signal peptide into a vector. I have designed 22bp primers + the 5 bps of the restriction enzymes. However, for all of them I am getting lots of inespecific amplification, I did it in silico and it seemed fine. I don't know if I should get longer primers and make sure there are 60 bps between each one or if I should just clone making overlapping primers by 15bps and creating primer dimers.
Thanks!
Hello Margarita,
Five (5) bp restriction enzyme site? Is that right? Secondly, when we design primers containing restriction enzyme sites, we also include 4-6 extra base pairs that serve as 'sitting sequences' so that restrction enzyme could bind to DNA stably and restrict it at the specified site.
For non-specific amplification, you may want to optimize your PCR reaction conditions. Try to do a gradient PCR to find out optimal annealing temperature.
Hi Margarita,
A few things:
If your primers have restriction enzyme sites engineered into them, make sure you also include a GC clamp on the terminal ends. This gives the restriction enzyme something to "grab" so it can more efficiently cut your engineered site. So, for example, if you are engineering a HindIII site you would want your Forward primer to be GGGCCCAAGCCT(then your target sequence of 18-22bp).
You will then need to optimize your PCR using these new primers. It doesn't happen all the time, but sometimes when you engineer RE sites onto primers, the parameters of the reaction conditions can change. You will want to ensure clean PCR amplification. From there, it's a gel purification, digestion of insert and vector, ligation and transformation.
Good luck!
lHi guys! Thanks for the response. For the restriction site, I add 4bps, usually something that lowers the Tm. For the PCR, I have tried gradient PCR, two step PCR, touchdown, DMSO, betaine, genomic DNA as a template, and I still get any unspecific amplification. I have also tried 3 different enzymes.
any ideas?
help!
Don't worry about TM when you are making primers that contain RE sites. You really need the GC clamp, + the RE site, +the 18bp target site. These primers usually fall in the range of a 65 to 70 TM, but that's okay.
I'm a little confused about the RE site you are using. Is it only a 4bp cutter?
About the template...genomic DNA. What concentration are you using? Also, are you sure that your primers don't span a very large intron? I am assuming you are doing a hot start PCR (TaqGOLD or an equivalent enzyme that is inactive until heated)?
I am using 50ngs and also I am working with bacteria, so I dont have to worry about introns
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