User:Etchevers/Notebook/Conference notes/2010/02/24

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Notes for Analyse Biorad ChemiDocs tutorial

Analyse Biorad ChemiDocs: Image Lab replaces Quantity One and it is downloadable.

New Protocol to acquire image but can also do other analyses during the capture eg. lanes and bands, molecular weight marker and size of bands.

First select application. All sorts of gels and blots are imagable. Chemiluminescence – with the least possible ethidium bromide. Colored eg. Sybr Gold, protein gel with Coomassie, silver… or a blot with colorimetric or alexa 488, Qdots or chemilum. Can add either no (chemi), or filter 1(most everything) or filter 2 (rare)

Pre-zoom uses "gel type Bio-rad ready gel" or manually specify gel size. Or, signal accumulation set-up = take images at a fixed interval and keep the one you like best. In seconds.

Display options – 65K gray levels, but if saturated, can highlight. Need it esp if quantifying.

Go to Run – can adjust place and zoom, live. Run protocol with parameters. No autocontrast – if signal not v intense, camera looks at all 65K levels of gray – you can adjust the transformation by reducing the gray spread. Pick an image and continue but only one is kept. Right-click could save all of them. If you save more than one, "cancel run" and re-open saved images. Image Lab format images can also be exported in other formats, starting with TIFF (raw image, will be required for a publication) or Displayed Image = JPG. Good for Word, PP.

For EtBr gels, pick either low or high sensitivity, also customizable.

For MW markers, add for each band; value = band size and description. Which lane (first or last) and regression – standard = linear (semi-log). Program starts from the smallest bands. You can also adjust and line up if some bands are crushed or off the gel. If you touch the gamma, curve deformation so falsifies the quantification. Low and high are linear and can be adjusted without loss of information proportions to one another.

A number of icons at top of image. Six analysis icons on the left. 2-4 are linked. Rotation of image is useful to line up bands. Right-click to rotate. It is possible to undo. Merge is useful when you don't have a chemiluminescent size marker for example. But you have to have images exactly the same size, before you crop.

Create Custom Application for a new marker – White epi illumination, no filter, gray, 2x2 and save. Then you can select the custom marker at the level of the gel imagine menu. Worry that too many of us would make too many custom markers. But these merges you can not use for the quantification, you can save the image for the size and the image will have a new name.

Display Gel Options is after analysis (intensities, size).

Lanes and Bands – can do an adjust when the gel or blot image is a little deformed. Enable Substraction – Disk size 10 (as smaller, removes more noise). Go into top icon "Lane Profile" to see what is being removed. Lane by lane. This is good for density calculation for the bands. (eg. compare 400 to 2000 or 0 to 1600).

Use Detect Bands and then you can add and delete or adjust the automatic band-finding feature. Lane Profile is also the area under each peak. Every one is modifiable manually.

can only put one molecular weight standard. Linear or point-to-point often (semi-log). The latter can help for high MW or extremes that are a little off the curve.

File-Display-Export Image or shortcut = Snapshot (eg with overlays).

Then you can make a Lane and Band table that tells you all the sizes and intensities of the different bands in the lanes. This table can be exported to an Excel table eg. or just kept as a CSV.

Quantification of bands in Quantity Tools – either relative or absolute, if you specify relative to the size/weight marker and the standard curve.

Annotation Tools.

Can pick the volume tools to label particular bands. This will add volumes to the Analysis Table – initial and w/o background-removed. Need a reference volume, first. And a background zone. This will yield in the table, a relative quantification.

Tutorial is present on the computer itself, can make a "report".

  • Heather 06:17, 24 February 2010 (EST):