Thursday, July 8, 2010

Take3 Tips and Tricks for Micro-volume Quantification

The Take3 Multi-Volume plate has proven to be a versatile, well adopted product since its launch last year. Applications include native 260/280 nm absorbance micro-volume quantification of DNA and proteins as well as more sensitive quantification with fluorogenic reagents such as PicoGreen for dsDNA quantification.

Here are a few of the important considerations to keep in mind when working with the Take3 plate.

1. Clean slides ensure great data! Here’s a quick way to check the cleanliness of the slides: Put 2-3 ┬ÁL of deionized, distilled H2O (ddH2O) on each spot and perform the blanking measurement with the Take3 interface in Gen5.
Use the “Use blank average” option in Take3 PreferencesCalculation and set the Validation Limit (%CV) to between 3 and 5 (whatever you are more comfortable with, the default is 10):

Read the plate. A clean plate should easily meet the limit. In the Applications Lab at BioTek Instruments, we routinely achieve CVs below 3% after thorough cleaning.

2. When working with extremely high concentrations of protein it may be necessary to clean the slide with a wetted wipe to remove any residual, precipitated protein. (This is most noticeable as streaking on the slide when wiping with a dry wipe). DNA does not appear to present this type of behavior even at very high concentrations.

3. Use a high quality multi-channel pipettor when possible to transfer samples, standards or reagents. This will help to eliminate some of the errors associated with multiple pipetting steps. Additionally, when working with any type of reaction chemistry on the Take3 Plate, the reagent addition should be done last and as quickly as possible to ensure equivalent kinetics across samples.

4. Gen5’s Take3 interface provides quick and easy nucleic acid and protein quant protocols, but the standard Gen5 protocol can also be used to define any number of micro-volume assays that would typically be performed in a microplate. The Take3 plate type is available in the Gen5 Plate database and can be selected to define a micro-volume protocol. Pathlength data can be easily entered into the data reduction steps using the values provided with each serialized plate.

5. Use replicate samples when possible. Replicate sample measurements can be used to identify flyers and ensure accurate results.

6. Change tips between transfers. The most accurate measurements will require consistent transfer volumes of the sample, standard or reagent. This is particularly important for assays that rely on reaction based chemistries. We have found that changing the tips often will help minimize pipetting errors.

It is best to treat the Take3 as you would any low volume transfer. Use the best pipettor for the task, preferably a recently calibrated 2 uL or 0.5-10 uL pipettor when possible. The use of low-adhesion tips can also improve the accuracy of transfer.

If you have further comments or questions or have developed your own assay for use on the Take3 plate we would like to hear them.


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