Prep for electroelution experiment 3-21-13

2015-01-13

azim58 - Prep for electroelution experiment 3-21-13

plan electroelution of DNA
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-dialysis membrane for electroelution of DNA
-http://humgen.wustl.edu/hdk_lab_manual/rdm/rdm9.html
--SPECTRA/POR dialysis tubing (VWR, B, F 10mm x 50 ft., cat.#132645) and
clips
---this product corresponds to a molecular weight of 6000 to 8000

-D-Tube Electroelution Accessory Kit
--they use Mini, Midi, or Maxi membranes with DNA (found in manual here:
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http://www.emdmillipore.com/life-science-research/d-tube-electroelution-acc
essory-kit/EMD_BIO-71511/p_C3Cb.s1OuKYAAAEjKBp9.zLX?attachments=USP

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-smallest cutoffs for Mini, Midi, and Maxi are 6, 3.5, and 3.5

-dialysis in our lab
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We have 1,000 MWCO tubing (Spectra/Por CE (Cellulose Ester) Membrane
131090
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http://www.spectrumlabs.com/dialysis/BiotechTubing.html?Pn=131090;
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It looks like this tubing should work for me

<protocol>
-basic notes about protocol (from here:
http://humgen.wustl.edu/hdk_lab_manual/rdm/rdm9.html)
-minimize exposure to UV (use prep (long form) of UV to cut out the DNA
-Cut a piece of dialysis tubing approximately 3 cm longer than the gel
slice and clip one end. Gently push gel slice into open end and down to
the clip. Add some buffer
-Place gel slices parallel to the electrodes and fill the electrophoresis
box with buffer (again, same as original gel) until all tubing is
submerged.
-Electroelute at 80-100 volts for 2-5 hours, longer for large fragments.
The movement of the DNA can be monitored with a hand held UV source
(long-wavelength).
-DNA may stick to dialysis tubing after current flow. Remove the gel
piece from the dialysis tubing. Reverse the electrodes and run for 15 s
to 2 min to dislodge the DNA from the membrane surface.
</protocol>

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