Mohsen,

Most of the time, proteins such as restriction enzyme bind to the DNA and the DNA migrate poorly (or get struck) on the gel. Add some SDS (0.1%) or Sarkosyl (0.1%) and then run the gel, you should not have DNA migration problem.

Is your PCR performed on genomic DNA? Maybe you used quite an excess of it...?

When DNA does not run well into the gel is that it is not well clean from proteins, although this should not be the case with a PCR product and a digestion, it could be your gDNA prep.

Other possibility, are you sure the comb used for the gel was clean enough? Not touched by hands or dirty gloves? You might have had keratin or dust on it...

Sorry I can't see the gel too well, did you have the same problem with all your samples? If so, maybe there's a difference in concentration between the buffer in the tank and the buffer used to make the gel?

Thanks a lot . As can be seen I didn't have the same problem with all my samples

1-check the grade of your agarose with samples you run before. If they look ok so the problem is not from your gel.

2-Always perform positive control and negative control. Digest a sample with KpnIeven it is not cut by KpnI.

3-Mae sure of the ratio of buffer to the total volume of the sample 10uL of buffer to 100 uL sample. measure DNA concentration of your sample before adding the restriction enzyme you may put 1uL for the first hour then one more uL in the second hour of digestion and please make sure of the optimum temperature

I added some SDS (0.1%)  and then run the gel,but i saw just a smear without a defined band.

Hi Mohsen,

1. See the Point 5 of this attached handbook ( "Troubleshooting Guide for DNA Electrophoresis" ) for 'DNA remains in the gel'.

2. They suggest: Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load, if add SDS. (See the yellow highlight).

I have the same problem too but it happens when i do repcr, not pcr. my pcr products are always ok but when i do repcr, bright bands stick in the wells. please let me know if you overcome your problem.

This file might be useful for everyone who has problem in this fields.