Hi everybody,
I would like to ask you if you have some suggestions to resolve my problem which is unreproducible measuring of RNA concentration. After RNA isolation I measured its concentration in water (for injection supposed to be RNAse free) in ratio 1:100. For measuring I am using Eppendorf Biophotometer..all settings are ok (I tried it on the two same machines in different institutes).
For example for one sample I get these results:- 460 subsequently 1320, 830 or 2nd sample 5288, 1132, 1212, 1793... ug/ml ...(measured independently)... the same cuvette was used.
thanks for advice in advance
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3 replies to this topic
#2
weimin42
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Posted 14 December 2004 - 11:32 PM
I measured the concentration of RNA for many times, in fact, although the variation between eplicates existed, but not so great. The reason may be your RNA were not dissolved completely into water, so uneven RNA lead to unstable result. It is better to dilute RNA by RNase-free water (pH>7.5)or TE buffer(pH=8.0).
Good luck!!
Good luck!!
#3
badcell
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Posted 15 December 2004 - 04:39 AM
Hi. I agree with weimin42, that it looks like your RNA is not well resuspended so you get erratic OD readings. I incubate the samples for 10 min at 55ºC after resuspension- I also use injection-quality water. This heating step really helps to redissolve the RNA pellet, specially in concentrated samples like yours, although it could be a problem if your sample is contaminated with RNAses.
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#4
kean_guy
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Posted 16 December 2004 - 12:11 PM
I used the same machine.
Here a few tips:
1. Once you've placed the cuvette into the Biophotometer slot, don't remove it during blank and sample readings. This will be your only cuvette for blank and sample readings. Rinse by pipetting sterile H2O in and out of the cuvette after each reading (but don't remove the cuvette from the slot at any time).
2. Avoid bubble formation inside the cuvette when you place the water or RNA solution. So pipette RNA solution or water into cuvette slowly and remove bubbles with pipette tips.
3. After each RNA solution reading, rinse the cuvette and add sterile H2O (blank), then press "read". The reading should be 0 (zero) ug/ml. If otherwise then re-blank and reread the RNA solution.
Good luck
Here a few tips:
1. Once you've placed the cuvette into the Biophotometer slot, don't remove it during blank and sample readings. This will be your only cuvette for blank and sample readings. Rinse by pipetting sterile H2O in and out of the cuvette after each reading (but don't remove the cuvette from the slot at any time).
2. Avoid bubble formation inside the cuvette when you place the water or RNA solution. So pipette RNA solution or water into cuvette slowly and remove bubbles with pipette tips.
3. After each RNA solution reading, rinse the cuvette and add sterile H2O (blank), then press "read". The reading should be 0 (zero) ug/ml. If otherwise then re-blank and reread the RNA solution.
Good luck
