I am working with urea-polyacrylamide denaturing gel for p32 lebelled oligonucleotide reaction with some drug in presence of topoisomerase IB in TBE 1X. The image of the gel is not so clear, does it depend on the concentration of TBE buffer or polyacrylamide or something else?
I too was working with radioactive gels recently. From my experience, one could improve the quality of the gels by considering following factors. Although they seem quite insignificant, you can implement them and check.
1. The choice of the comb that you use to prepare the gel plays some role. Try different sized combs and you'll see the difference. At one point you'll end up getting sharp bands.
2. Expose your gel to the film for longer times. I could observe sharper bands on doing this.
3. Sometimes, the gel is not well in contact with the film due to some problems in the cassette. Check that.
4. Allow your gel to dry for sufficient time. Wet gels will give smeary bands.
Hope these points help.
Best Regards,
Sagar Sridhara
Thanks Sagar,
well first 3 points i generally check, the 4th one i will try, another thing i found during my experiment, that if i run the gel in 2X TBE (instead of 1X) buffer then clarity of the image get bit enhanced.
Regards
Prafulla
Hey,
That is nice to hear. I hope it works out for you.
Thanks,
Sagar Sridhara
I did these kinds of gels for manual DNA sequencing long ago. Check you're ratio of acrylamide:bis in your gels. 19:1 is good for short denatured DNA oligomers. 29:1 is good for longer ones. If you're using 29:1 and resolution is poor, switch to 19:1.
- Badges
- Science topic
- Similar topics
- Gels