I ran an agarose gel electrophoresis with genomic CHO DNA samples. Some of the samples have been treated with RNase (lanes 2, 4 and 6 - picture attached). There is a band stuck in every well - what could that be? In RNase lanes 2, 4 and 6 this band is much less prominent suggesting the content was degraded by the enzyme. Does that mean there is RNA stuck in wells? How come if there are RNA smears seen lower in the gel...Thanks!
Hi Ana,
The following link may help you:
https://www.researchgate.net/post/Why_is_my_DNA_stuck_in_my_well_of_an_agarose_gel#:~:text=The%20presence%20of%20DNA%20binding,an%20atypical%20band%20migration%20pattern.
"It happens due to one of these reasons;
1-Poorly-formed gel wells:
Remove the gel comb only after complete polymerization of the gel. Pour the buffer onto the gel immediately. Rinse the wells with electrophoresis buffer to remove urea from denaturing polyacrylamide gels prior to loading the sample
2-Excess DNA loaded:
Follow the recommendations for loading described in the certificate of analysis of the DNA ladders/markers (~0.1-0.2 µg per 1mm gel lane width) or in the Table 9.6 on p.445. If possible load the same quantity of the sample.
3- contamination of the DNA sample
4- gel shift affect:
The presence of DNA binding proteins in the sample, such as ligases, phosphatases or restriction enzymes may alter DNA migration in the gel and cause the DNA to remain in the gel wells. Lambda DNA or other DNA with long complementary overhangs may anneal resulting in an atypical band migration pattern. To eliminate these effects, use 6X DNA Loading Dye & SDS Solution which is supplemented with 1% SDS to eliminate DNA-protein interactions and to prevent annealing of DNA molecules via long cohesive ends. Always heat these samples with SDS at 65°C for 10 min, chill on ice, spin down and load."
Hello Ana Marentič
The DNA has not been extracted properly. The DNA sample shows RNA as well as protein contamination. RNA molecules are lighter than DNA. So, the RNA migrates faster than the DNA and DNA migrates faster than the protein.
So, if RNA contamination is present, one would see a faint and smeary RNA band below the genomic DNA. You use RNase in lanes 2, 4 and 6, so the RNA smear has become much less prominent which means the RNA has been degraded by the enzyme.
The band stuck in every well is not RNA but could be because of the presence of protein contamination in the sample which may cause the DNA to remain in the gel wells.
So, you could try a proteinase K digestion at 55°C O/N and then a phenol chloroform extraction.
Hope this helps.
Best.
Hi
Could indicate contamination (Can be gDNA so as Malcolm said, try proteinase K digestion) or you are maybe loading too much sample into the wells. I usually load about 4uL
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