Jun 13, 2016

Public workspaceDot Blot Preparation

DOI
dx.doi.org/10.17504/protocols.io.etibeke
  • David Dunigan and Irina Agarkova1
  • 1The University of Nebraska-Lincoln
  • VERVE Net
Irina Agarkova
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DOI: dx.doi.org/10.17504/protocols.io.etibeke
Protocol Citation: David Dunigan and Irina Agarkova 2016. Dot Blot Preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.etibeke
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: March 31, 2016
Last Modified: March 27, 2018
Protocol Integer ID: 2634
Guidelines
Materials:

1.) host and viral DNAs
2.) 1X TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA)
3.) 1 M NaOH
4.) 1 M Acetic acid
5.) nylon membrane, cut to 12.3 X16 cm (2 sheets)
6.) Whatman 3MM filter paper, cut 12.3X16 cm (2 sheets)
7.) SSC solutions: 1X, 5X, 20X
       20X SSC (1000 mL):
  • 175.3 g NaCl
  • 88.2 g sodium citrate
  • adjust pH to 7.0 with NaOH
8.) 96-well assay plates
9.) hybridot dot blot apparatus and pump

Note: Load the membranes so that when scanned, the second blot of the set may be placed under the first, i.e., load as the following diagram shows:
DNA dilutions
DNA dilutions
Prepare DNAs in 1.2 µg/48.6 µl concentrations.
Distribute 97.2 µl of each to 96-well plates in the same pattern as the 1.0 µg samples will be loaded onto the membrane.
Make 3 serial 1/2 dilutions with each DNA sample (48.6 µl into 48.6 µl 1XTE).
Discard 48.6 µl from the final dilution.
Add 5.4 µl of 1 M NaOH to each sample.
Cover plates with parafilm.
Incubate at 37°C, 30 min.
Duration00:30:00
Add 6.0 µl of 1 M acetic acid to each sample.
Chill on ice.
Membrane preparation
Membrane preparation
Wet the membrane in 1X SSC ≥60 min.
Duration01:00:00
On the hybridot manifold, place a piece of Whatman 3MM filter paper and wet with 20X SSC.
Roll air pockets out.
Mount the membrane on top of the 3MM paper (centered).
Roll air pockets out.
Wash the membrane with 5X SSC (ca. 25 ml) with vacuum filtration.
Screw the top of the hybridot manifold in place, then shut off the vacuum pump.
Add 200 µl of 5X SSC to each well.
Add DNAs (50 µl each) to the wells.
Turn on the vacuum pump and draw samples onto the membrane.
Wash each well with 400 µl of 5X SSC (2X).
Remove membrane and crosslink under UV light (200 mJ).
Store the membranes wrapped in plastic at 4°C.
Note
Note: Load the membranes so that when scanned, the second blot of the set may be placed under the first, i.e., load as the diagram in guidelines shows.