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In our hands, polylysine slides require a pre-hybridization step to lower background fluorescence.
Pre-hyb solution:
25 ml 20X SSC
1 ml 10% SDS
1 gram BSA
74 ml dH2O
Dissolve BSA thoroughly
Filter through a 0.2 micron filter
Pre-warm at 45° C in coplin jar for at least 2 hours
Slide Preparation:
- Mark location of spots and label slide with a diamond tip pencil
- Rehydrate in a humid chamber of 0.3 M potassium phosphate for 30 min.
- UV crosslink in Sratalinker on 1,000 energy setting = 1,000 X100 ujoules
- Wash 30 sec. in 0.1% SDS
- Boil in water 3 min.
- Snap cool in cold dH2O
- Place in 45° C pre-hyb solution
- Pre-hyb at 45° C for 1 hour
- Wash by hand dipping slides several times in a large volume (500 ml) of room temperature dH2O
- Shake slides dry
- Set up hybridization immediately
Other protocols:
- Staining Slides with Sybr Green HTML | PDF
- cDNA Synthesis and Labeling HTML | PDF
- Labeling Amino-linked Oligos HTML | PDF
- Pre-hybridizing Polylysine Slides HTML | PDF
- Pre-hybridizing CodeLink Slides HTML | PDF
- Hybridization and Washing HTML | PDF
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