The constitution of digest are as follows:
water - 15ul
Buffer - 2.5ul
pcr - 7ul
enzyme - 0.5ul
I have also tried several other adjustment still the same result..
Please can someone tell the right voltage and time for running the gel.
Hi
this link can help you
https://www.researchgate.net/post/What_causes_ghost_bands_in_restriction_enzyme_digests
Hi there!
1. You need to know the concentration of DNA you are using for digestion. As already mentioned, there is a correlation between the amount of DNA to be digested and the reaction volume (50uL per microgram approximately). You can look up the given link.
2. If your band is of low molecular weight then it will take up less EtBr in the agarose gel and you'll get faint bands which doesn't mean your digestion hasn't worked, but it is mere visualisation problem. You see that in the lower molecular weight bands (100 and 250bp bands in a 1Kb ladder).
3. Load your equal amount of undigested PCR product alongside your digest and see if there is any difference. If you can provide with a gel picture it'll be be very helpful to pin-point the problem.
4. Its difficult to suggest electrophoresis conditions without knowing the band size. If your desired band is small, run a lower percentage gel and give it a short run.
Hope it helps!
https://www.researchgate.net/post/How_to_decide_the_volume_of_restriction_digestion_reaction
If I understood your question, you don't have problems with ghost bands? Just with their intensity after running the gel?
I think that voltage and time for running the gel won't matter much for band intensity, only with their "resolution". If you want to have nice bands you should run the gel on low voltage for longer time in gel with higher agarose percentage.
However it will not help much if you have PCR problems.
Are the faint bands correct size after digestion?
What is the loading volume of your sample? It Should always be less than the hold volume of the loading well? Otherwise, sample full volume may not be loaded onto the gel and Some sample may go into the electrophoresis tank.
The other thing is What is the concentration of Agarose you are using? If you are visualizing low molecular weight fragments ( less than 500 bp) you need to use high concentration gel. such as 1.2% or higher. The voltage of the electrophoresis apparatus also matters to a certain extent, I typically run at 60V.
Please try also the following
1. analyzing your PCR sample also on an another electrophoresis apparatus.
2. Load undigested PCR sample along with the digested.
3. Estimate the amount of the PCR product using the standards you have before you start to digest. because to visualize clearly you need at least 20 -50 ng DNA per band. so suppose you get 04 bands in the digestion, at least 100 -200 ng of PCR product must be digested and loaded to visualize them clearly.
Have you run your PCR product on a gel before the digest? I feel that there is not enough DNA in your sample could be a reason. Failure of your PCR reaction or the quality of your isolated DNA does matter. Have you measured the concentration of your DNA isolates to begin with.
If you are getting faint band of desired product then you can do the following changes
1. Double the volume of your PCR product.
2. Increase time of restriction digestion like u can perform the same digestion for 2 hr , 4 hr and 6 hr interval and see the change in intensity of band after running gel and the select the desired time. Also if your enzyme don't show star activity u can perform the digestion overnight too.
3. if the first two steps don't help then increase the enzyme units.
if you are getting faint band of undesired product then the reason can be partial digestion to remove the faint band do the step number 2 only.
Also if the bands of your DNA ladder are sharp, then their is no need to change The voltage and time of gel running u r asking for.
but Yes if the ladder band is not sharp then the problem can be in your gel preparation or etbr amount
Hope it will help you
You have to consider the time of incubation. Some enzyme has star activity that can cut your DNA in several parts. Also check the concentration (unit per activity) to digest a fragment of DNA. I
Gel Purify the PCR product before restriction digestion. Run the undigested PCR product of equivalent concentration in a parallel well while doing electrophoresis.
I believe u r doing it for cloning purpose.
PCR salt ingredients I the buffer may hamper restriction, so gel purification may help
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