Hello,every one
I cleaned up my RNA samples with DNase1 , I made RT-pcr to make sure that sample become free from genomic DNA ,but i had in my gel result a smear in the distal part of 2 lanes as photo show below
lane 1: 100 bp ladder.
lane 2: control positive PCR to make sure that the pcr mix work good .
lane 3: 2hr time interval negative rt-pcr.
lane 4: 4hr time interval negative rt-pcr.
lane 5: 8hr time interval negative rt-pcr.
lane 6: 12hr time interval negative rt-pcr.
lane 7: 18hr time interval negative rt-pcr.
lane 8: 24hr time interval negative rt-pcr.
lane 9: NTC for pcr
thanks every one
Looks like a low molecular weight smear. You can use this guide from biorad to start with and try to fix the problem.
http://www.bio-rad.com/en-us/applications-technologies/pcr-troubleshooting
I would also reccommend that you used aliquoted reagents - that way you can discard any old/contaminated aliquot, use filter tips that should be changed with every sample that you load on your agarose gels, use high quality water. These are some general tips. Good luck!
https://www.thermofisher.com/in/en/home/references/ambion-tech-support/rna-isolation/general-articles/ten-ways-to-improve-your-rna-isolation.html
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