GE RPN2510E User Manual download pdf (Page 13)

RPN2510EUM Chapter 9 Rev C 06/2007 13

Symptom

1. Membrane curling up in

hybridization bottle

2. High background

3. Weak signal

4. Patchy backgrounds

5. High background around edge of

membrane

Cause

1.1. Incorrect orientation of bottle in

rotisserie

2.1. Probe concentration too high

2.2. Unincorporated

P nucleotides not

removed

2.3. Insufficient blocking

2.4. Insufficient washing

3.1. No transfer from gel to membrane

3.2. Probe not labeled

3.3. Probe not denatured

3.4. Low specific activity of probe

3.5. Washes too stringent

4.1. Hybridization buffer or wash

solution not evenly covering

membrane

4.2. Damaged membrane

5.1. Damaged membrane

Remedy

1.1. Ensure membrane is rolled up in

the same direction as the bottle is

rotating (see section 7.2)

2.1. Reduce probe concentration

2.2. Remove unincorporated

nucleotides

2.3. Use recommended hybridization

buffer or extend prehybridization

time

2.4. Increase number of buffer changes

during the washing stage or

increase stringency of final wash

3.1. Load extra lanes with control DNA.

Transfer can be checked by

restaining gel with ethidium

bromide. If large DNA fragments

are detected poorly, use a

depurination step (0.25 M HCI)

3.2. Check probe labelling before

hybridization. See protocol in probe

labelling booklet

3.3. Boil probe for 5 minutes before

adding to hybridization buffer

3.4. Review labelling conditions

3.5. Increase buffer salt concentration

and decrease temperature

4.1. Increase volume of hybridization

wash or solutions or use mesh (see

section 7.1. step 4)

4.2. Handle membrane carefully with

forceps

5.1. Use clean scissors or a sharp

scalpel to cut membrane

9. Troubleshooting guide

This section briefly summarizes some of the potential problems encountered during membrane hybridizations. More

complete troubleshooting guides are supplied in the pack leaflet that accompany each product.

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