Hi guys,
i tried to amplify a drug resistant gene from a plasmid(around 3kb). The gel photo I got shoe two prominent bands of really big size, and a faint band(~1.3kb), which is the size of the gene.
Is it possible that this is due to too much template added?
I have 300ng of plasmid Dan in 50ul pcr mixture.
Thanks!
Peggy
Hi Peggy,
300 ng of plasmidic DNA is way to much for a PCR reaction. I would recommend to bring the template down to a concentration of 10 to 50 pg in the final PCR reaction. You could also try to play around with the number of PCR cycles, for example, starting with 35 rounds. I wonder what Tm your oligos have and the timing of amplification.
Cheers!
Hi Peggy,
300 ng of plasmidic DNA is way to much for a PCR reaction. I would recommend to bring the template down to a concentration of 10 to 50 pg in the final PCR reaction. You could also try to play around with the number of PCR cycles, for example, starting with 35 rounds. I wonder what Tm your oligos have and the timing of amplification.
Cheers!
Hi Miguel,
I first tried diluting my plasmid (template) to 60ng and 30ng respectively, and do the PCR again. However, I found only the prominent bands of big sizes on my gel this time. (the band that I want to amplify disappear....)
(lane1: 60ng template; lane2: 30ng template; lane3: marker)
The Tm that indicated on the primer sheets are both around 70C. But the PCR reaction I did, I use 60C as annealing temperature.
Maybe I should try lower annealing temperature? More diluted template?
Peggy
I also want to add that:
The primers that I used to amplify the drug resistant gene have extra 60bp homology sequence to the flanking region of a yeast gene (I want to amplify this homology seq-drug resistant gene-homology and then do yeast transformation to generate mutant)
-->This might lead to inefficient PCR?
Primer dimer showed up when I diluted the template
-->Is the inefficient PCR really due to too much template in the reaction?
A weird thing I observed is that the sizes of the two prominent bands in the original PCR rxn are different from the PCR rxn with diluted templates (as you can compare to the marker)
I'm now trying to do gel extraction of the first PCR product (just cut the band of right size and elute it)...but I still wonder why is it that the drug resistant gene not the prominent band?
Thanks!
Peggy
Hi Pei-Ching Huang,
If you can, elute the faint band(~1.3kb) from the gel. and do PCR for the same with same primers and same PCR conditions for at least 32 cycles.
I hope that helps!
Hi Peggy,
Drug resistant gene you want to amplify from plasmid, is a gene insert ? If so, you can try PCR using plasmid primers (universal). It will gives your desired gene product along with some part of plasmid, but certainly it will help you to avoid non-specific amplification. From plasmid primer product, you can try another PCR using gene specific primer. If your gel protocol is working, then no need to apply this.
Thanks
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