rRNA & polysaccharide contamination...

[fibonacci]

does anyone know how many ribosomal RNA yeast have???

usually when i extract the RNA, i only get 2 intact bands (28S & 18S)..

BUT somehow, when i do the RNA extraction, i will get 3 bands...

there are another band 'upper' than the 28S rRNA...(but it is not genomic DNA because i run the RNA with 1kb ladder and the size of the 'upper band' is around 4.5kb...)

does anyone know about the band????

please help me..

 

when i measure the absorbance of my RNA, usually i will get a good reading of A260/280 which is around 1.8-2.0....but A260/230, usually i will get around 1.40-1.70...

how can i get rid of the polysaccharide contamination?????

any suggestion????

 

p/s - i do the RNA extraction by using TRIZOL from Invitrogen....

[CharonY]

The easy bit first:

the A250/A230 ratio suggests guanidine isothicyanate contamination (from the Trizol). To clean that up add another precipitation/ethanol washing step.

Regarding the additional band: it is hard to judge w/o really seeing the gel. However, note that of course chromosomal DNA, especially if sheared, will not have the correct size as indicated by the marker. The marker is only accurate for completely linearized DNA. Also, did you use a denaturing gel and how long was the DNAse treatment?

For better troubleshooting the complete protocol as well as a picture would be beneficial.