Bio-rad Zeta-Probe Membranes User Manual

Zeta-Probe®Blotting MembranesInstruction ManualBio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547LIT234 Rev CLIT234C.qxd 11/3/2003 11

4.2 Formamide ProtocolPrehybridization1. Seal the blotted Zeta-Probe membrane inside a heat-sealableplastic bag. Prepare the following solution for p

2x SSPE1% (w/v) SDS0.5% (w/v) BLOTTO10% (w/v) dextran sulfate0.5 mg/ml nonhomologous carrier DNA4.4 Oligonucleotide Protocol6Prehybridization1. Seal

2. Cut one corner of the bag, remove the prehybridization solution, and replace it with the same buffer.3. Add probe, then seal the open corner (tak

Section 6Probe Stripping and RehybridizationIf reprobing is desired, do not allow the Zeta-Probe membrane to drybetween hybridizations.The Zeta-Probe

Problem3. High background observedthroughout membrane on autora-diographSolutionThe major contributors to background areunincorporated label, small r

Problem7. No autoradiograph signalSolution1. After transfer, stain the gel to check thattransfer was complete. If not, increasetransfer time and/or

10% BLOTTO g/100 mlNonfat powdered milk 100.2% sodium azide 0.2Store at 4°C20% SDS MW g/L20% sodium dodecyl sulfate 288.38 200Heat to 65°C to get into

Section 10Ordering InformationCatalog Number Product Description162-0153 Zeta-Probe Membranes, 9 x 12 cm, 15 sheets162-0154 Zeta-Probe Membranes, 10 x

Table of ContentsSection 1 Introduction ...1Section 2 Nucleic Acid Blotting Protocols ...

Section 1IntroductionZeta-Probe blotting membranes are nylon membranes which haveunique binding and handling properties that make them ideally suitedf

diagonally and aligning the opposite corners with the gel corners.Then lower the Zeta-Probe membrane onto the gel.6. Cut two pieces of 3MM paper to t

7. Cut two pieces of 3MM exactly to the gel size. Wet a sheet ofprecut 3MM paper in water and place it on the Zeta-Probe membrane/gel stack, then rep

Soak one fiber pad by squeezing it while it is sub-merged in 0.5x transfer buffer. Lay the soaked pad onthe open gel holder. Soak a piece of thick fil

7. Disconnect the vacuum, disassemble the apparatus, and rinsethe membrane briefly in 2x SSC. UV-crosslink the DNA to themembrane or vacuum dry the b

Alternative hybridization protocols are necessary when probe lengthsvary outside this recommended range (refer to Oligonucleotide Protocol, Section 4.

The Tmis decreased approximately 1.5°C for every 1% decrease inhomology.10, 11The Tmis decreased as the fragment length of the probe decreases;the app