In this scheme, a random polypeptide library encoded by a plasmid vector is analyzed by PCR. The library is obtained by transforming bacteria with the product of a ligation reaction, as described in Scheme 2, followed by amplification and purification of the plasmid using standard procedures. This experiment was derived from PCR Laboratory Guide (2nd edition) by Seed Kang and Qu Lijia.

Operation method

Quality control experiments of a randomized peptide DNA library by PCR

Materials and Instruments

ddH20 Vent buffer Vent DNA polymerase dNTP primers Plasmid DNA
Reagents and equipment for agarose gel electrophoresis Thermocycler

Move

I. Materials

1. Buffers, solutions and reagents

_ddH20

2. enzymes and enzyme buffers

10X Vent buffer

Vent DNA Polymerase

3. Nucleic acids and oligonucleotides

dNTP, 20 mmol/L each

primers that can anneal to vector sequences flanking the cloning site of the synthesized DNA library

Plasmid DNA (see step 1 below)

4. Specialized equipment

Reagents and equipment for agarose gel electrophoresis, including ethidium bromide (see step 5)

Thermocycler

II. Methods

1. After amplification in bacteria, perform the following reactions with plasmids prepared from randomized peptide libraries.

10~50ng of plasmid DNA

50pmol of each primer

5ul of 10X Vent buffer

dATP, dCTP, dTTP and dGTP, 0.5ul each

1U of Vent DNA Polymerase

Vent DNA Polymerase, 0.5ul of each of _dTTP and dGTP.

The various plasmid vectors below should be reacted separately:

Expression vector without library DNA insertion

DNA library mixture

Small amount of DNA, prepared from a single bacterial clone of the DNA library

2. Preheat the mixture for 20 s at 94°C in a thermal cycler.

3. Perform 30 rounds of PCR cycles according to the following parameters.

94°C10s

55°C10s (or appropriate annealing temperature with primers)

72°C15s

Extend the extension time of the last cycle to 30s.

4. Cool to 4°C.

5. Take 25ul of each reaction product, run a 4% agarose gel electrophoresis and analyze with ethidium bromide staining (Sam-brook and Russell 2001).

Analysis

In the example shown in Figure 26-4, analysis of the PCR product, using a 12-nucleotide random library mixture as a template, showed that



Analysis of the PCR product from the 12-nucleotide random library mixture as a template showed that most of the library contained inserts of the correct size (Figure 26-4, lane 2). Furthermore, no PCR product corresponding to the original vector was detected (compare Figure 26-4, lanes 1 and 2). PCR analysis of individual clones from randomly selected libraries confirmed that most of the inserts were of the correct size and that there was no contamination of the original vector in the libraries (Figure 26-4, lanes 3-20). Analysis of the DNA sequences confirmed that these PCR products were the correct members of the 12-nucleotide random library (data not shown). Therefore, PCR analysis of the reaction intermediates (Scheme 2, Figure 26-3) estimated that a high quality final library had been obtained (Figure 26-4).

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