PCR-based DNA blotting experiments

Summary

For high-throughput screening, it is recommended to use 96- or 384-well PCR plates available from several thermal cycler manufacturers, or to use individual tubes. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.

Operation method

PCR-based DNA blotting experiments

Principle

dNTP, PCR buffer, polymerase mix, nested primers, LB-aminobenzyl liquid medium, NaOH, SSC, Tris-HCl, microtitration plates for PCR products, nylon membranes, UV cross-linking device, 96-well or 384-well replicator, thermal cycler, PCR tubes or plates

Materials and Instruments

dNTP PCR buffers Polymerase mix Nested primers LB-aminobenzyl liquid medium NaOH SSC Tris-HCl PCR products
Microtitre plates Nylon membranes UV cross-linking device 96-well or 384-well replicator Thermocycler PCR tubes or plates

Move

Phase 1: Amplification of DNA insert by PCR

I. Materials

1. Buffers, solutions and reagents

dNTP solution (contains all 4 dNTPs, 10 mmol/L each)

2. enzymes and enzyme buffers

10X PCR buffer (40 mmol/L Tricine-KOH, pH 9.2 at 22°C, 3.5 mmol/L Magnesium Acetate, 10 mmol/L Potassium Acetate, 75 mg/ml BSA, or as supplied by the manufacturer)

Polymerase Mix, 50X (Advantage2, Clontech, or equivalent)

3. Nucleic acids and oligonucleotides

Nested primers NPundefined

Nested Primers NP2undefined

~undefinedOr, primers flanking the insertion site on the vector can also be used for PCR amplification of the inserted fragment.

4. Culture medium

LB-Aminobenzyl liquid medium

Prepare 1L of medium by dissolving 10 g of bacterial tryptone, 5 g of bacterial yeast extract and 10 g of NaCl in 950 ml of non-ionized water and adjusting the pH to 7.0 with 5 mol/L NaOH, then make up the volume to 1L with non-ionized water and autoclave the medium for 20 min at 151bf/in2(llbf/in2=6894.76Pa). After autoclaving, the medium was cooled and ampicillin was added to a final concentration of 50ug/ml.

5. Specialized equipment

Thermal cycler

At this stage, PCR is performed on the plate and previous accuracy must not be required, thus making the use of mineral oil unnecessary.

PCR tubes or reaction plates

6. Additional reagents

Reagents and equipment needed for agarose gel electrophoresis

7. Cells and Tissues

cDNA library, present in suitable strains, grown as colonies on LB agar plates

II. Methods

1. 96 or 384 white bacterial colonies were randomly picked from the library.

2. On a PCR plate, each colony was incubated with 150ul of LB-aminobenzyl medium at 37°C for at least 4 h (or overnight) with gentle shaking.

3. Prepare a master mix for 100 or 400 PCRs.



4. Dispense 19ul of Body Mix into each well of each tube or reaction plate.

5. Take 1ul of each bacterial culture (see step 2 above) and add it to each well of each PCR tube or PCR plate containing the Master Mix.

6. Perform PCR in a thermal cycler using the following conditions without mineral oil.



7. 4ul of each reaction is analyzed on a 2% agarose gel.

Phase 2: Spot Blot Analysis

I. Materials

1. buffers, solutions and reagents

0.6 mol/L NaOH (freshly prepared, or newly diluted from a concentrated storage solution)

SSC, 20X (1L formulation: 175.3 g NaCl and 88.2 g sodium citrate, pH 7.0)

0.5 mol/L Tris-HCl, pH 7.5

2. Nucleic acids and oligonucleotides

PCR products

3. specialized equipment

Microtitre plates

Nylon membrane

UV crosslinking device (e.g. Stratagene's UVStratalinker), or 68°C oven

96-well or 384-well replicator

II. Methods

1. Take 5ul of each PCR product and mix it with 5ul of 0.6mol/L NaOH.

2. From each mixture, apply 1 to 2 ul to a nylon membrane. This step can be performed by dipping a 96- or 384-well replicator into the appropriate wells of the microtitre plate used for PCR amplification and dispensing onto a dry nylon membrane. Spot at least 2 identical membranes for hybridization with the forward and reverse subtraction probes (Scheme 1, Phase 3). It is highly recommended to spot 4 identical nylon membranes. 2 to hybridize to forward and reverse ablated DNA, and 2 to hybridize to the initial cDNA or genomic DNA.

3. Neutralize with 0.5 mol/L Tris-HCl (pH 7.5) for 2~4 min, and wash with 2XSSC.

4. Fix the DNA on the membrane with a UV cross-linking device (e.g. Stratagene's UVStratalinker) and bake at 68°C for 4 hours.

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