I have been trying to isolate RNA from S.aureus by using Qiagen RNeasy Mini Kit. Cells are disrupted by silica beads with FastPrep Homogenizer before the isolation. After purification A260/280 ratio is always in optimal range (~2.0) but I could not obtain A260/230 ratio higher than 1 (optimal range is 2.0-2.2). What could be the problem? Do you have any suggestion to fix this?
Thank you.
Hi Cemile,
One guess is that buffer RLT that contains a chaotropic salt absorbing at 230 nm is not completely washed away during the RNA isolation process.
1) Do you do the two RPE washes before eluting RNA off the column as described in the protocol? The RPE washes get rid of residual salts remaining on the column.
2) After the RPE washes, do you centrifuge your columns for one minute to completely get rid of residual ethanol etc?
Best,
Nesli
Cemile Selin Aksoy
You can increase your cell concentration. You may use the RNA purification kit. You must be careful during RNA isolation due to contamination and degradation problem of RNA.
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