Improved Recovery for 3-10kb DNA Size Selection

BluePippin users, we have a new cassette definition that will appear in the Cassette Definition Set 10 (CD10) that we feel merits some further detail. This definition is named “0.75% DF 3 – 10kb Marker S1 – Improved Recovery

What is it?
This definition improves the sample recovery for customers using BLF7510 Marker S1 cassette kits for DNA size selection, for size ranges between 3- 10 kb. The original cassette definition for this range, “0.75% DF 3 – 10kb Marker S1” uses pulsed-field during elution (size selection), and we have subsequently found that direct-current elution improves recovery.

Who should use it?
This is now the recommended cassette definition to use for size-selecting between 3-10kb. However, if you have optimized a protocol using the current version you may want continue to use it (it will not be deleted as a cassette definition option) if you do not wish to re-optimize.

Also note that since the BluePippin system does not allow for simultaneous pulsed-field and direct-current operation, lanes that are being separated using pulsed-field will temporarily be halted during every elution process. This makes the total run times lengths dependent on the elution times (and size selection targets), and therefore more difficult to estimate and can add up to 40 minutes to a cassette run.

How much improved is the recovery?
Our validation of the change in the cassette definition shows up to a 2-fold increase in recovery. As you may know, for collections of large fragments (on 0.75% agarose cassettes) we provide Tween solution which we find improves recovery if one employs a post-collection Tween rinse. Considered together, here are some results from our study. We started with a 5 ug of restriction digested DNA, and 8kb “Tight” collections:

DC elution image for blog

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