I want to fuse 2 genes (4kbp each gene). I amplified both of them by PCR adding an overlapping region of 27 bp and gel extracted them.
First thing that I don't like is that I can't quantify the amount of DNA obtained of each gene because I always get contamination at 230nm (measured by nanodrop). I've tried several gel extraction kits ( Quiagen, Omega and GE Healthcare). Nevertheless, if I run 5ul of the PCR results into a gel I get clear intense bands of my products.
Then I moved on to the overlap PCR and I always get smears.
I use the forward primer of gene A and the reverse primer of gene B (1ul and 4ul each were tested from a 10mM stock). I've tried different DNA volumes of each template gene (1ul each, 2ul each and 5 ul each). I've also tried KOD and Vent polymerase and different annealing temperatures (since the lowest Tm of the primer is 66.8 I've tried annealing at 65, 62 and 55 ºC). All of these conditions were tested with 25 cycles of Overlap PCR rx.
Any idea of why I'm getting a smear and what can I do?
The smear may be due to too much input DNA in your second PCR. You should never put pure PCR products into a new PCR mix, because it is way too much. If you have nice bands on an agarose gel, I would dilute your samples at least 1:100 and then use 2ul.
As for your nanodrop measurements, this is totally normal. Chemicals used to dissolve the agarose absorb in the UV, and you can hardly get rid of them. To quantify your samples after the first PCR, compare your bands intensity from an agarose gel with the ladder. You can check on the suppliers website the amount of nanograms in each band of the ladder you use.
The smear may be due to too much input DNA in your second PCR. You should never put pure PCR products into a new PCR mix, because it is way too much. If you have nice bands on an agarose gel, I would dilute your samples at least 1:100 and then use 2ul.
As for your nanodrop measurements, this is totally normal. Chemicals used to dissolve the agarose absorb in the UV, and you can hardly get rid of them. To quantify your samples after the first PCR, compare your bands intensity from an agarose gel with the ladder. You can check on the suppliers website the amount of nanograms in each band of the ladder you use.
It may be due to high quantity of input DNA, so first you measure the actual quantity using Real Time PCR based any available kits, the go ahead.
This means that you have a high concentration of genomic DNA and thus resulting in a smear. Use DNA at a final concentration of 20ng and it should fix the smear problem.
Hi, Adrià Alcaide Jiménez and all others.
In order to be able to give you good tips, I would ask you to send us a few more data.
For instance, could you send the data from your PCR protocol? Any gel pic showing/confirming you got your product amplified and containing the overlap when compared to the product without overlap?
One tip is, If you have the product with overlap and without any extra T at the end (proofreading enzyme), you may let them ligate by chance putting your reaction into the tube with buffer, water, salts and both PCR products and heating it to 95ºC and then cycle decrease the temp by 1ºC to the annealing temp. Then, You can check If you find (by gel visualization), a band with 8KB, gel purify and use it, in a diluted amount as your template for PCR.
Hope it helps.
Hi all, thanks for your help. I solved the problem. The nanodrop was broken and the reads were wrong. I wasn’t putting enough template to run the pcr.
- Badges
- Science method