Bioanalyzer: Difference between revisions
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* wash with clean water for 3min immediately after the run has ended (in our experience the electrodes in the lid will quickly deteriorate otherwise) | * wash with clean water for 3min immediately after the run has ended (in our experience the electrodes in the lid will quickly deteriorate otherwise) | ||
* to export data as PDF you have to ''print''; (PDF option checked will save file, PDF unchecked will print on paper) | * to export data as PDF you have to ''print''; (PDF option checked will save file, PDF unchecked will print on paper) | ||
== Troubleshooting == | |||
A few typical mistakes occur once in a while: | |||
* small RNA marker is not properly (often the next peak is selected => fragments size mislabelled) | |||
: cleak peak table, a tab at the bottom of the window; lower marker can be manually set here | |||
* 18S, 28S not properly assigned (=> RIN number incorrect) | |||
== See also == | == See also == | ||
Revision as of 09:03, 10 July 2009
| back to protocols | ||
The Bioanalyzer is a chip-based capillary electrophoresis machine to analyse RNA, DNA, and protein. It is produced by Agilent and widely used, among other things, in RNA quality control measurements before downstream experiments like microarrays.
Protocol for Bioanalyzer RNA nano chip
preparation of material
- 12 samples per chip
- quantitative range 25–500 ng/μl
- quantitation accuracy 20%CV (for ladder as sample)
cleaning, gel preparation (start 40min before experiement)
- take out filtered gel aliquot and fluorescent dye from fridge next door 30min ahead of time (1 gel aliquot tube enough for 2 chips)
- take out ladder from fridge upstairs
- vortex dye 10s and spin down
- mix one tube gel aliquot with 1µl of dye
- vortex, centrifuge 15000g +-20% for 10min
- wash electrodes 1min RNase ZAP, 3min water (pipette into 1 well, will spread)
prepare chip for measurement
- take out new RNA chip (pico or nano) from drawer below microarray machine and put into station
- fill 9µl gel into dark circle G well (gel reservoir)
- press down plunger and wait 30sec (gel moves through channels)
- after 30sec release silver trigger (should jump up well above 0.3, meaning seal was tight)
- wait 5sec, pull plunger up all the way
- open priming station, add 9µl gel to light circle G wells
- 5µl of marker into all sample wells and ladder well, bottom right (contains small marker to align plots)
- 1µl of sample per well, add 1µl of water or replicates into free wells (6µl required per well for machine to run properly)
- 1µl of ladder into ladder well
- vortex in holder for 1min and put into machine
start the run
- start 2100 expert software
- choose programme for kit: DNA/RNA pico/RNA nano and material: total RNA/mRNA, prokaryotic/eukaryotic
- start run
wash, export data
- typical RNA nano run takes 25min
- wash with clean water for 3min immediately after the run has ended (in our experience the electrodes in the lid will quickly deteriorate otherwise)
- to export data as PDF you have to print; (PDF option checked will save file, PDF unchecked will print on paper)
Troubleshooting
A few typical mistakes occur once in a while:
- small RNA marker is not properly (often the next peak is selected => fragments size mislabelled)
- cleak peak table, a tab at the bottom of the window; lower marker can be manually set here
- 18S, 28S not properly assigned (=> RIN number incorrect)
