Apogee 11056017 Gebruiksaanwijzing - Pagina 7

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2.3 APPLYING THE MEMBRANE AND BLOTTING PADS

Nitrocellulose and PVDF (positively-charged nylon) membranes are commonly available and both work

well.

1. Make any needed identifying marks in soft pencil or preferred marker on a piece of the dry,

precut membrane.

Note: Handle the membrane with gloved fingers or smooth-tipped (non-serrated) forceps to

prevent oils and proteins on fingers from blocking efficient transfer.

Note: PVDF membranes require pre-treatment by soaking in methanol for 1 to 2 minutes followed

by soaking in transfer buffer for 5 minutes.

2. Wet the membrane by floating it on distilled water for a few moments and then immerse it in

transfer buffer for 1 to 2 minutes.

3. Align the membrane carefully on the gel taking care not to create any air pockets.

Note: Do not allow the membrane to touch the wicking sheet during transfer. If it does, a 'short

circuit' will occur and buffer will move around rather than through the gel.

4. Place the tray insert, with the wicking sheet, gel and membrane on it, into the buffer tray with

the ends of the wicking sheets extending into the tray.

5. Place a stack of blotting pads on top of the membrane, and the gel restrainer over the blotting

pads (Figure 5). As with the membrane, be careful not to let the blotting pads touch the wicking

sheet. To ensure even contact through the blotting stack, place a weight on top of the gel

restrainer.

Figure 5: Configuration for capillary transfer

2.4 CAPILLARY TRANSFER

1. Fill the buffer tray approximately half full with transfer buffer. To ensure even capillary flow,

make sure the ends of the wicking sheets are equally immersed.

2. Allow from 4 to 24 hours for complete capillary transfer depending upon the size of the DNA or

RNA fragments and the percentage and thickness of the gel.

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