Hi all,
We have repeated this multiplex PCR multiple times to amplify 5 genes using previously published primers and primer concentrations. We have not been able to get distinct bands upon gel electrophoresis in a 1.5% gel with 1X TBE run at 80V. All the correct amplicons are present, but the bands smear one into the next. Any suggestions on what we can modify to improve the appearance of our gels?
Thank you!
Kayla
hope this link is useful
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3153144/
regards
Check this link maybe helpful
http://www.aun.edu.eg/gembc/ahmed%20%20Gaballa/PCR-trouble%20shooting.pdf
regards
Hello Kayla Lea Bieser,
this is a typical case of electrophoresis bands "smiling" (also known as curved bands). Solution could be following:
1.) Decrease voltage (80 to 60 or 50 V)
2.) Run gel at 4°C or in chilled buffer
3.) Decrease concentration of TBE (1x to 0,5x)
4.) Try to use TAE instead TBE
Hope this help :-)
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