I am using freshly puchased ambion trizol reagent. i tried using the protocol provided but not getting intact bands. i thought problem can be due to other reagent and changed chloroform and isopropanol but problem persist as before. sample was harvested on month back and stored at -80 C and seems well. What could be the probable cause? Any suggestion would be highly appreciable.
Was your material stored in RNAlater? Your starting material can be degraded.
Hello Ashok,
Try to isolate RNA from some fresh (or, practice) samples. I doubt it is the water. Yes, the sample should have been stored in RNAlater, but, 1 month in -80 C is an alright time to still get good yield of RNA.
Good luck,
Venk
samples with high protein content always make problems in PCR, So I suggest to make another extraction for the same sample using lower volume of sample to reduse protein contamination
Ashok, i agree with Venkatesh sir. once try to isolate RNA with fresh sample. if it is ok, problem is in your sample, otherwise use all freshly DEPC treated material for RNA isolation and avoid contamination to get good quality RNA.
Hello Ashok, Mahesh,
Also, just wanted to mention, try and use other kits as well, if possible.
I work with miRNAs, for example, and we used 4 different kits, efore we decided on one.
Most suppliers will be happy to give "free samples".
Good luck,
Venk
Hi Ashok..
Problem may be with the sample if it undergone through freezing, thawing or if it is very old sample.. other possibilities are... your chemicals and solvents, u r using like chloroform, isoamylalcohol or ethanol, that may get contaminated. Better u try with fresh samples n newly opened solvents.
once we were facing the same problem..n we came to know that RNA was ok but the problem was with GTC, which we use in preparation of RNA gels..
All d best...
Dear All
Thank you for your precious attention.
there was problem with DEPC treated water. At ethanol washing step i diluted 100% ethanol to 70% with RNase free water and i got intact band with good yield.
once again thank you
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