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BluePippin Operations Manual 460013 Rev F
6-1
6
Sample Preparation
Input Sample Characteristics
When running the BluePippin, characteristics of input DNA can affect separation
resolution and efficiency of product recovery. The following general guidelines should
be followed:
•
Ionic strength:
The ionic strength of the sample should be lower than the
ionic strength of the buffer (80mM monovalent ions). High salt concentrations
can result in slower than expected DNA mobility.
•
Protein in the sample:
DNA-binding proteins such as ligases or polymerases
can affect the mobility of fragments during separation. Proteins can also reduce
DNA recovery from the elution module by increasing the binding of DNA to the
ultrafiltration membrane at the back of the elution module. For best results,
samples should be de-proteinized prior to loading whenever possible.
•
Input DNA size distribution:
A knowledge of the input size distribution is
obviously important to program accurate size selection settings. BluePippin
cassettes are calibrated using the Agilent Bioanalyzer to evaluate input and
product sizes, and so, for best results, input size distributions should be
evaluated using the Bioanalyzer. For low concentration samples, the Agilent HS
chip is very useful.
Preparing DNA Samples for the BluePippin
1.
Bring
DNA sample up to 30μl with TE.
2.
Bring internal standard mix (or loading solution if using an external marker) to
room temperature.
3.
For each sample, combine 30μl of DNA sample with 10μl of internal standard mix
(or loading solution).
4.
Mix samples thoroughly (vortex mixer). Briefly centrifuge to collect.
Recommended sample Load Guidelines
Maximum Load:
5
g
sheared genomic DNA
Minimum Load:
15 ng
, sheared genomic DNA