For the gel electrophoresis a 2 % agarose gel is made up (dimensions 20x20cm, 3-4mm thick). The gel is run for 4hrs at 85V. As running buffer 0.5x TBE is used.
A band pattern of 5-10 different bands (200-600bp) is expected depending on the sample. Some of the bands can be very similar in size and very close to each other. The gel image is not clear at all and the different bands cannot be distinguished.
Would high resolution agarose be a solution?
Image of your gel would be better to understand.i think ,you give more time to gel run,by that time your PCR product become out of the gel area.Gel chamber buffer volume is also a factor,better to give buffer 2 mili-miter over the gel level.From my daily experience with (HLA-Typing gel run),i have seen that,greater buffer volume over the gel, causing faint band or low resolution band,.
So,from my point of view,solution may includes
1.give less time at high voltage like 30 min at 150 Volt.
2.Adjust added buffer level during gel load.(not more than 2 or 3 mm) over the gel.
3.Check you Primer annealing temperature or Tm value of primer,for ensuring that you are getting right PCR product you wanted .Good Luck.
We have used 3% NuSieve agarose + 1% regular high melting temperature agarose in 0.5x TBE buffer. This should work fine for the band sizes in which you are interested.
Running the gel for long time may cause lateral diffusion of your sample too. If you think you have two bands of similar sizes. You can purify them by cutting from 2% gel and later of 2.4 or 2.6%. You should take care of the viscosity of buffer that you are using.
I have the same problem. What improve a little for my case was use TBE 0.5X, gel 2 or 3% low melting for 2h - 85V. I realize, like Sarang Mahajan tell, more than 2h I can't have better separation between the bands and cause diffusion in sample. I just got very good results using PAGE. I would like to know how to improve resolution in this case too.
Image of your gel would be better to understand.i think ,you give more time to gel run,by that time your PCR product become out of the gel area.Gel chamber buffer volume is also a factor,better to give buffer 2 mili-miter over the gel level.From my daily experience with (HLA-Typing gel run),i have seen that,greater buffer volume over the gel, causing faint band or low resolution band,.
So,from my point of view,solution may includes
1.give less time at high voltage like 30 min at 150 Volt.
2.Adjust added buffer level during gel load.(not more than 2 or 3 mm) over the gel.
3.Check you Primer annealing temperature or Tm value of primer,for ensuring that you are getting right PCR product you wanted .Good Luck.
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