TotalBLOT NORTHERN Kit

Directions for Use

NORTHERN BLOTTING ONTO A NYLON MEMBRANE WITH HIGH-SALT BUFFER

• Agarose -10g (Code 0710)

• 10X MOPS -2 x 500 ml (Code E526)

• 37% Formaldehyde -200 ml (Code 0493)

• Formamide -10 ml (Code 0606)

• 20X SSC -4L (Code 0804)

• TotalBLOT+ Nylon membranes (optional)

• Whatman 3MM filter paper sheets

• Plastic wrap (e.g., Saran Wrap)

A. Gel Preparation:

For a 100 ml gel, dissolve 1g agarose in 72 mL diH2O by heating gently (in microwave or on hot-plate). After solution has cooled to about 65°C, add 18 mL 37% formaldehyde and 10 mL 10X MOPS. Cast gel and allow to polymerize.

B. Sample Preparation:

• Bring the volume of the RNA samples to be analyzed to 11 L with DEPC-treated diH2O. Add to this 5 L 10X MOPS, 9 L 37% formaldehyde and 25 L formamide. (Alternatively, a stock of 100L 10X MOPS, 180 L 37% formaldehyde and 500 L formamide can be prepared and 39 L of the stock solution added to the RNA samples; Store stock frozen.)

• Mix briefly and incubate at 65°C for 5 min. Quick cool on ice.

• Add to each sample 5 L of a 10X dye mix (or 10 L of a 5X dye mix).

C. Gel run:

Load the samples and apply a voltage of 5V/cm until the bromphenol blue dye has migrated approximately two-thirds of the length of the gel. Stain the gel in 0.5 g/ml ethidium bromide and destain in diH2O. Complete destaining may require an overnight incubation in diH2O. Photograph the gel.

D. Equilibrate the gel and membrane:

Soak the gel in 20X SSC for 10-15 min. Cut a piece of membrane slightly smaller than the gel to be blotted. Wet the membrane in 20X SSC.

E. Upward Capillary Transfer:

Prepare the transfer pyramid as follows:

a. Place a solid support (usually glass or plastic plate) over a reservoir (so that the plate spans the reservoir) filled with 20X SSC.

b. Prepare a wick by cutting a strip of 3MM filter paper long enough to be placed over the glass plate allowing the ends to be submerged in the transfer buffer. The wick must also be cut slightly wider than the gel to be blotted. Saturate the wick with 20X SSC.

c. Place the gel on the saturated wick. Remove bubbles by rolling pipette over the surface. Place four strips of plastic wrap over the edges of the gel.

This is to prevent the buffer from short-circuiting and flowing around the gel. If properly sealed, the buffer will flow through (rather than around) the gel.

d. Cut a piece of TotalBLOT+ membrane the same size as the gel to be blotted. Wet the membrane in diH2O and equilibrate in 20X SSC for 5-10 minutes.

e. Place the wetted membrane on the surface of the gel and remove any bubbles by rolling a glass pipette over the membrane.

f. Flood the surface of the membrane with 20X SSC. Cut 3 to 5 sheets of Whatman 3MM paper to the same size as the membrane and place these on top of the membrane.

g. Cut paper towels to the same size as the membrane and stack these on top of the Whatman 3MM papers to a height of about 5 cm.

h. Lay a glass plate on top of the structure and place a weight on top to hold everything in place. Leave overnight.

The weight should be sufficient to compress the paper towels to ensure good contact throughout the stack. Excessive weight, however, will crush the gel and retard transfer. Ensure that the weight is evenly balanced. Unbalanced weight will result in unequal transfer of nucleic acids. An overnight transfer is sufficient for most purposes. Extend the transfer time if the gel concentration is > 1%, or transfer of fragments > 20 kb is desired. Make sure that the reservoir of 20X SSC does not run dry during the transfer.

F. Completing the transfer:

• Remove the paper towels and filter papers and recover the membrane. Mark (in pencil) the position of the wells on the membrane and notch one corner of the membrane to ensure proper orientation.

• Rinse the membrane in 2X SSC, then place it on a sheet of Whatman 3MM paper and allow to dry. Store at room temperature.

TotalBLOT+ membranes do not require baking or UV crosslinking

Montluçon

tel (33) (0) 4 70 03 88 55 

fax(33)(0) 4 70 03 82 60

 http://www.interchim.com

Vos contacts:

Etienne Boireau - Olivier Cadas - Olivier Alméras

hot line  33 (0) 4 70 03 73 06