Hi researchers,
Please see the attached image. This is a gel image of a 8kb PCR product from genomic.Each two adjacent lanes are the same sample from a 50ul PCR system. I use 6 pairs of primers to do the PCR.
The amount of template DNA for all of them is the same, 200ng/sample. Annealing temperature is different, according to each primer pair. All the rest reagents are the same.
Can see from the gel, some sample got bright bands and some are faint. My question is, will the bright bands be complete the PCR product? In other words, the brighter bands are also "wider', whether these is junk DNA in these "wider" bands? Or they are certainly my aim PCR product. They are "wider" just because in that condition, I get more product?
Thanks!
Hello Shuaichen:
Is the gel image from an End Point PCR? Then if it is I think that probably and due to for example the Plateu Effect what you are getting in your wider band is not the PCR product you want to get. Efficiency and error increase in PCR as long as you have more PCR cycles and thus your primer hybridize to sequences others than your target.
The first four lanes seem fine to me and perhaps you are close to the best annealing temperature for those primer sets.
Also I would try higher annealing temperatures or lower annealing times for the other lanes or maybe shorter PCR cycles (eg. 30 instead of 40).
A great idea to know if you are getting the product you are looking for in the others lanes would be to sequence the product.
Greetings.
Hi Rodrigo,
Thanks for your reply! This is a 35-cycle product. Can you explain further about what is Plateu Effect in PCR reaction?
Hello Shuaichen:
You are welcome.
As cycle increase the PCR reduces its efficiency. In the early cycles efficiency is theorethically close to 2.0 but as PCR cycling increase it is reduced. In this context, it affects enzyme and also the other components of the PCR. Therefore if you have more cycles the PCR product of your interest target does not increase including inespecific products.
I think 35 cycles is fine but maybe try reducing to 30 or also the other options I previously mentioned.
From the gel image bands 1 to 4 look good. It looks like there are nonspecific binding of the primers giving you nonspecific bands in the wider ones. So I would suggest you to do a gradient PCR from +5 to -5 of the Tm of your primers (preferably between 50 to 70), to find the right binding temperature for the primer to the template. Also, I think you need to reduce the amount of DNA in your samples with broader bands.
High DNA concentrations give fat bands because there is a limit to how much DNA you can squeeze into a given volume of agarose - among other things - the physical chemistry is quite complex.
If you want to know if your fat bands are pure, dilute 1:10 and 1:100. Then you should see a nice sharp band.
Note also that electrophoretic mobility is slightly affected by DNA concentration.
I agree with Andrew, your problem is probably due to the high concentration of DNA you use initially. Try using the dilutions mentioned above, and if you still get these bands you might change other parameters. In addition to the Tm and the cycles you could try reducing the amount of primers used, or even adjust the MgCl2 concentration. Caution, change one parameter at once!!
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