Our band capture cassettes, for use on the BluePippin, are designed to automatically collect a band or fragment of DNA from a sample and remove unwanted DNA and/or primer artifacts. We’ve provided users with some flexibility to manipulate the capture parameters, outlined below. Unlike the ethidium-containing cassettes used on the Pippin Prep, we require users to add a DNA binding dye, Midori Green, prior to use so that the system can detect your sample DNA. Band Capture cassettes part numbers are BLG7510 for 2 kb – 10 kb bands, and BDG1510 for 250 bp – 1.5 kb bands.
What is Midori Green?
Midori Green is a safer alternative to ethidium bromide that was developed by Nippon Genetics Europe, GmbH. Its fluorescent properties are easily detected by the BluePippin’s fluorescein-compatible optics.
You can read about Midori Green here, it’s a great product.
How to use band capture cassettes
Midori Green will lose its intensity with prolonged storage at room temperature, so we ask users to store at 4oC and add it to our cassettes just prior to use. A small amount is added to the opposite end of the gel cassette (like ethidium, it electrophoreses in the opposite direction of DNA). In software, users enter a software base pair threshold value, somewhere between 80% and 90% of the anticipated band size. The system will collect the very next band it detects after that threshold has passed.
We also provide some simple tools to help users tweak their collections in the form of “expansion factors” . In the software protocols, these parameters are labeled “Start Exp” and “End Exp.”, and they are used to determine how close (or far away from) the DNA band the collection occurs. In “tight” mode collections of sheared DNA, the Pippin collects the narrowest distribution possible. The timing of these collections (“BP Start” and “BP End”) are uses as guidelines to determine the timing of band capture. This figure below shows the position of the collection timing, and where we’ve set the default “expansion factor” settings. We’ve widened the gap by increasing the base pair values by 10% on either end.
The figure below shows the parameters fields in a BluePippin protocol for a 10 kb band collection (band capture cassettes use “peak” mode). The “Start Exp” and “End Exp” values may be programmed to move the arrows in the figure above accordingly.
Sample loads over 5 ug/band will likely need protocol adjustments
The default settings should be fine for sample loads that are 5ug/band and below. Since higher loads have a higher DNA-to-dye ratio, they run faster, and the bands are typically broader. We rarely see the need to change the “Start Exp” value, but the “End Exp” typically requires a boost – we find that 1.50 ”End Exp” value will collect the widest band we’ve tested, but some method development may be required:. This figure illustrates the type of band broadening that occurs at higher sample loads.