260
WILLIAM M. STRAUSS
mine- and fluorescein-labeled probes or 0.6 ~g/ml of propidium iodide
(PI) for fluorescein-labeled probe.
It is recommended to analyze the slides immediately afterwards. Fluorescent signals can, however, be well retained -30°C for a few weeks.
Troubleshooting
Do not use polystyrene materials for handling or storing PNA.
Keep PNA in dd. H 2 0, not in neutral pH buffer that may cause PNA
precipitation at low temperatures.
Dissolve PNA in pre-warmed hybridization buffer and place PNA in
37-50°C for at least 15 min.
Use telomeric or centromeric repeat PNA as a positive control. If control signals are seen clearly.
Check the concentration of PNA by scanning at 260 (230-650) nm. Examine the second peak for labeled dye.
Make sure hybridization and washing procedures have been executed
properly. Lower hybridization temperature from 37°C to 30°C or even
room temperature or decrease formamide concentration from 70% to
40% for hybridization and washing, particularly for shorter PNA 12mer or is-mer or AT-rich sequences.
loss of PNA a.
b.
c.
No signals or a.
weaker signals
b.
c.
High background a. Too much PNA probe. Check PNA concentration.
b. Increase stringency ofhybridization and washing such as a higher concentration of formamide, increased washing temperature, and longer
time.
c. Add a blocking step in the procedure, simply by treating slides with
1x blocking solution at 37°C for 5 min, air dry, and go to hybridization
step.
d. During the process for making chromosomal slides, make sure any
medium (mainly serum) is completely washed away using 1x PBS.
WILLIAM M. STRAUSS
mine- and fluorescein-labeled probes or 0.6 ~g/ml of propidium iodide
(PI) for fluorescein-labeled probe.
It is recommended to analyze the slides immediately afterwards. Fluorescent signals can, however, be well retained -30°C for a few weeks.
Troubleshooting
Do not use polystyrene materials for handling or storing PNA.
Keep PNA in dd. H 2 0, not in neutral pH buffer that may cause PNA
precipitation at low temperatures.
Dissolve PNA in pre-warmed hybridization buffer and place PNA in
37-50°C for at least 15 min.
Use telomeric or centromeric repeat PNA as a positive control. If control signals are seen clearly.
Check the concentration of PNA by scanning at 260 (230-650) nm. Examine the second peak for labeled dye.
Make sure hybridization and washing procedures have been executed
properly. Lower hybridization temperature from 37°C to 30°C or even
room temperature or decrease formamide concentration from 70% to
40% for hybridization and washing, particularly for shorter PNA 12mer or is-mer or AT-rich sequences.
loss of PNA a.
b.
c.
No signals or a.
weaker signals
b.
c.
High background a. Too much PNA probe. Check PNA concentration.
b. Increase stringency ofhybridization and washing such as a higher concentration of formamide, increased washing temperature, and longer
time.
c. Add a blocking step in the procedure, simply by treating slides with
1x blocking solution at 37°C for 5 min, air dry, and go to hybridization
step.
d. During the process for making chromosomal slides, make sure any
medium (mainly serum) is completely washed away using 1x PBS.