Field of Science

How much washing is enough?

Today I'm testing my preps of beads with DNA bound to them by streptavidin-biotin linkages.  I want to know how much DNA is on the beads, and to be sure that this DNA is held by a streptavidin-biotin bond at one or both ends, rather than being passively trapped by other bead-DNA linkages.

I had four preps I'm made previously, two to EcoRI-cut DNA and two to XhoI-cut DNA; the average fragment sizes are about 6 kb and about 12 kb respectively.  I also have two new preps of a mixture of both DNAs, bound to magnetic Dynabeads (either  2.8 µ M280 beads or 1 µ T1 beads.  All of the preps were washed three times with about 100:1 volumes of TE, and resuspended in 100 µl of TE.  DNA concentrations have only been measured for one of the EcoRI and one of the XhoI preps.

The goal is to find out whether the intervening incubation and an additional thorough washing (rotating overnight in 10 volumes of TE) will remove more DNA from the beads.

So last night I measured the volume of each bead+DNA prep and took a 5 µl sample, to be assayed today for DNA concentration using the Picogreen assay.  The beads+DNA I did assay had about 250 ng DNA/ml, so this 5 µl was chosen to give enough sensitivity without using up too much of my preps.  Then I added 9 volumes of TE to each prep and put the tubes on the roller wheel in the 37°C incubator overnight.  Now I am going to pellet the beads (centrifuge or magnet), remove the supernatant, and resuspend the beads at their original concentration (= original volume - 5 µl).  Then I'll take 5 µl of the beads+DNA and 25 µl of each supernatant for Picogreen assay.

For assay standards I have lambda DNA at 1000, 100, 10 and 1 ng/µl, and lambda DNA mixed with beads at known concentrations (to check that the beads don't interfere with the assay).

I hope to find that most of the DNA is still associated with the beads. 

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