I have been extracting RNA for 5 months, I should have planned to use 5'RACE and 3'race to get the coding strand.
The RNA I extracted is always seriously degraded. mRNA is not complete, that's hard for me to do 5'RACE and 3'RACE.
I thank bio-rad good for rna extraction.
http://www.bio-rad.com/en-ch/category/rna-isolation
There are several commercially available kits that should be suitable. However, it is important when extracting nucleic acids from fungi to also ensure that you are adequately destroying fungal cell walls which can be quite tough. I would recommend adding extra processing steps, such as additional bead-beating or other mechanical disruption steps to increase your yield. Of course, it is helpful to be very diligent in eliminating RNAses as well.
Thank you all!
Though the imagine of RNA which was observed by agarose gelelectrophoresis didn't show good integrity, next experiment was still being carried out. The results showed 5' and 3' RACE seemed to be successful. I’m going to use TA-cloning to further determine the result whether is correct.
The following is the imagine of 5' and 3' RACE PCR.
5' RACE failed.
Trace remaining DNA contaminated the experiment.
I have to extract total RNA again.
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