subtractive hybridization experiment

Summary

The experiment introduces subtractive hybridization, which is derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

subtractive hybridization experiment

Materials and Instruments

Dilution Buffers Hybridization Buffer Storage Solution Triethylammonium Bromide Mineral Oil Nucleic Acids and Oligonucleotides
Thermal cycler

Move

I. Materials

1. Buffers, solutions and reagents

Dilution buffer (20 mmol/L HEPES>HCl, pH 8.3, 50 mmol/LNaCl,0.2 mmol/L EDTA)

4X hybridization buffer storage solution: 4 mol/L NaCl,200 mmol/L HEPES, pH 8.3, 4 mmol/L hexadecane

Triethylammonium Bromide (CTAB). Final 1X hybridization buffer, diluted with dilution buffer.

Mineral oil

2. Nucleic acids and oligonucleotides

Detector 1-1 for ligating Ad1 (Step 8 of Phase 4 of Program 1)

Detector 1-2 linked to Ad2R (Scheme 1, Phase 4, Step 8)

Driver DNA (Scheme 1, Phase 3, Step 5)

Rsa I digested driver DNA (Scheme 1, Phase 3, Step 5)

3. Specialized equipment

Two thermal cyclers (see steps 11 and 12)

II. Methods

1. First hybridization

(1) Mix each tester sample with the reagents in the order listed below.

(2) Cover the sample with 1 drop of mineral oil and centrifuge briefly.

(3) Incubate the samples in a thermal cycler at 98°C for 1.5 min.

(4) Depending on the sample type, incubate the sample at 68°C for the time listed below and proceed immediately to the second hybridization.



This protocol uses 15 ng of ligated assay DNA and 450 ng of driver DNAa If different ablation efficiencies are desired, change the driver and

This protocol uses 15ng of ligated detection DNA and 450ng of driver DNAa The ratio of driver to detector can be varied if different ablation efficiencies are desired.

2. Second hybridization

(9) (No steps 5~8 in the original text.) Repeat the following steps for the driver DNA of each experiment.



Add the following reagents to a sterilized 0.5 ml (1ul) centrifuge tube.

(10) Take 1ul of the mixture, add it to a 0.5 ml centrifuge tube, and add 1 drop of mineral oil to cover.

(11) Incubate for 1.5 min at 98°C in a thermal cycler.

(12) Remove the freshly denatured driver centrifuge tube and immediately place it in another thermal cycler that has stabilized at 68°C.
The use of another thermal cycler (or another block) will ensure rapid cooling of the sample.

(13) Then, add the hybridized Samples 1-1 and 1-2 (from the first hybridization) in the same order.
This ensures that the two hybridized samples fit together in the case of freshly denatured driver DNA overdosage.

(14) Incubate the hybridization reaction solution at 68°C overnight.

(15) Add 100ul of Dilution Buffer and mix by pipetting.

(16) Incubate in a thermal cycler at 72°C for 7 min.

(17) Store the hybridization reaction solution at -20°C.

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