Science Gateway

Protocols: ES Cell Culture - Gene Targeting - Protocols

Transfer of DNA to GeneScreen Plus Using the Bio-Dot 
SF Apparatus


	1.	Cut one piece of GeneScreen Plus and two pieces of 
Bio-Dot Slot format filter paper (Bio-Rad; cat. #162-0161) to the 
exact size of the dot blot apparatus.

	2.	Place the membrane and pieces of filter paper in 0.4 M 
Tris-HCl pH 7.5; allow to soak for 30 minutes.

	3.	Prepare the samples in clean Eppendorf tubes as follows:

a.	Dissolve 10-20 ug of each DNA sample in 100 ul TE pH 8.0, total 
volume per sample.

b.	Add 5 ul of 5 N NaOH to each tube and denature @ room temp. for 
10 minutes.  (The solution will be 0.25 N with respect to NaOH.)

c.	Chill on ice and add 100 ul of 0.125X SSC to each tube 
(diluting the solution to 0.125 N NaOH).

	4.	Assemble the dot blot apparatus as follows 
(refer to Figure 1):

a.	From bottom to top, assemble the vacuum manifold, the gasket 
support plate, and the sealing gasket.

b.	Remove the two pieces of filter paper and the GeneScreen 
Plus from the Tris solution; blot to remove excess liquid.  
Assemble the pieces of filter paper on top of the sealing gasket, 
making sure that no air bubbles get trapped beneath.  Next, place 
the nylon membrane on top of the filter paper, removing any trapped 
bubbles.

c.	Secure the sample template on top of the nylon membrane 
using the sealing screws.  Tighten diagonally opposite screws at 
the same time to prevent wrinkling of the membrane.

	5.	Load the diluted DNA into the slots in an ordered 
array.  Add 200 ul of 0.125X SSC into those slots that do not have 
any sample.  Allow the solution to remain on the membrane without 
any suction for 30 minutes.

	6.	After 30 minutes, apply suction until all the wells 
are visibly dry, approximately 2 minutes.

	7.	Disconnect the suction and take the dot blot apparatus 
apart.  Carefully remove the membrane and place DNA-side up on a 
piece of Whatmann filter paper.

	8.	Allow the filter to dry @ room temp., cross-link the 
DNA to the membrane using the Stratalinker, and prehybridize in 
the usual manner.