TE 22 Mini Tank Transfer Unit

Overpacking the cassette, and then running the unit at temperatures in excess of 50 °C causes the cassettes to become bowed. Bowed cassettes will always pack improperly and cause diffuse or swirly band patterns on the membrane.

Transfer buffer temperatures must be kept below 50 °C. Cooling requires an external cooling water recirculator, such as the MultiTemp III.

Preventive action: "Proper cooling" does not mean using tap water in the base.

Plastic parts have become warped

1. Circulate only water or 50/50 water/ethylene glycol through the heat exchanger. Never introduce anti-freeze or organic solvent into any part of the instrument. Organic solvents will cause irreparable damage to the unit.

2. Buffer temperature should not exceed 50 °C. Prevent overheating by circulating coolant during the transfer and using only the recommended buffers. The transfer can be conducted in a cold room. Please note that circulating of coolant is still needed in a cold room. Overheating will cause irreparable damage to the unit.

Corrosion of electrode post (on electrode panel) or electrode socket (in lid)

Clean the corroded parts

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Check the power supply with another electrophoresis unit to see if the output is okay

Loose electrode post on electrode panel.

Fasten the electrode post

Blank areas on the membrane

1. Remove all trapped air pockets in the transfer stack assembly: assemble the stack while it is submerged in transfer buffer, gently press on each sponge as it is added to the stack, and roll a glass pipette or test tube over the membrane and gel to eliminate all air bubbles.

2. Reduce the stirring speed to prevent turbulence.

3. Process only one strip or membrane in each tray or cassette to prevent overlapping.

4. Use buffer with a lower ionic strength.

5. Check electrode continuity. During the transfer, a continuous stream of gas is released along the entire length of the electrodes. If bubbles do not form along the entire length of the electrode, replace the electrode.

6. If cassettes are bowed when empty, replace. Overpacking the cassette causes it to bow.

Grid pattern on membrane

Add extra sheets of blotting paper to increase the clearance between the cassette panel and the gel. Take care not to overstuff the cassette; the gel should be held firmly and evenly between the sponges, but not so tightly that it is squeezed.

Molecules do not migrate out of gel

1. Increase the field strength.

2. Increase transfer period. (Try doubling it.)

3. Do not use staining or fixing agents on the gel before transfer.

4. Use a thinner gel.

5. Reduce the gel acrylamide concentration.

6. Check that the buffer pH is close to the intended pH. Most buffers should not be titrated; make fresh buffer.

7 Use 3.5 mM SDS (0.1%) in the transfer buffer.

8. Avoid including methanol in the transfer buffer or reduce the amount to the absolute minimum.

9. Use reagent-grade chemicals.

10. Increase the length of time Southern blots are depurinated.

11. Increase the net charge on the protein by changing to a transfer buffer with a different pH. Lower pH (<6-7) increases the positive charge on proteins; higher pH (>6-7) increases the negative charge on proteins.

Power supply diode/fuse has blown by high current

Unit requires servicing

Preventive action: Lower power supply current limit

Bubble is present in stack or above bottom electrode, caused by excessive current

Lower power supply current limit

All components were not soaked in transfer buffer

Soak all components in transfer buffer

Stack is dry or partially dry

Re-wet stack

Safety interlock is defective

Unit requires servicing

1. Transfer immediately after electrophoretic separation. If equilibrating before the transfer, shorten or eliminate the equilibration time or move the gel to the cold room during equilibration.

2. If transfer buffer contains methanol (³ 10%), equilibrate the gel in transfer buffer for 30 minutes to allow it to shrink before assembling the stack. Note: Because methanol causes the gel to shrink slightly, large molecules may migrate more slowly.

3. Take care that the gel is held firmly against the membrane and that it does not shift once contact is made.

4. If excess heating occurs during the transfer, lower the temperature of the cooling fluid in the heat exchanger.

5. Check that the preferred binding surface of the membrane (if any) contacts the gel.

6. Evaluate the unit. Electrode panels may be warped from excessive heat. Warped electrodes give warped electric fields, resulting in diffuse/smeared transfers.

7. Buffer depletion can change the pH in the transfer stack, and have a negative effect on the trans

Distorted or swirled transfers due to loose stacks

Increase the number of sponges in the stack

Post has been exposed to buffers.

Post must be kept clean and dry to avoid corrosion. If corrosion is observed wipe and dry the area. In extreme cases, the banana plug posts and/or the high-voltage leads may need to be replaced.

Blown fuse in protection circuit.

Replace fuse.

Chemical parameters are not optimal.

1. Fix or crosslink the molecule onto the membrane according to the requirements of the nucleic acid, protein, or membrane type.

2. Prepare protein transfer buffer without SDS.

3. Verify the optimal amount of methanol required for the membrane type and check the buffer solution. Add 10 - 20% methanol to the transfer buffer to enhance binding to nitrocellulose.

Membrane parameters are not optimal.

1. Wear gloves when handling membranes.

2. Store membranes properly. Protect them from temperature extremes and direct sunlight.

3. If proteins pass through the selected membrane, try a different type or one with a smaller pore size (0.10 - 0.20 µm).

4. If different proteins may be migrating in opposite directions, place a membrane on both sides of the gel.

5. If the sample load may be exceeding the capacity of the binding surface area, apply two membranes. If "blow through" occurs, reduce the sample load.

Blotting paper too large

The blotting paper and membrane must be the same size as the gel or 1– 2 mm smaller. Larger sizes will provide an electrical path for current to bypass the gel solution.

Different sizes and/or net charges of proteins

Different proteins will transfer at different rates depending on size and net charge.

Stack is over-wetted

Buffer should not be oozing out of the stack when the top assembly is placed on it, nor should there be pools of buffer on the bottom electrode. Please note: No contact or path should be available that allows current to bypass the gel.

Trapped air pockets between the gel and the membrane

Remove trapped air pockets between the gel and the membrane during stack assembly.

Presence of gas

Lower the current setting to minimize the amount of gas produced by the electrolysis of buffer.

Buffer has too high ionic strength and hence generated too much current

Use buffer with a lower ionic strength. Please note: Do not back-titrate transfer buffers