Biotech Glossary | Bioinformatics | Lab Protocol | Notes | Malaysia University |
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Nco I and Not I Restriction Digestion of the scFv Gene Repertoires |
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| Contributor: |
The Laboratories of Andrew Bradbury at Los Alamos National Laboratory and James D. Marks University of California, San Francisco |
| Overview |
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| In this protocol, the PCR products recreating the scFv fragment and the vector encoding the PIII protein (pHEN 1) are digested with restriction enzymes and ligated together to create a library of phage scFv antibodies. The scFv fragments have previously been joined together in a separate ligation reaction that also appends the necessary restriction sites (see Protocol ID#2199). Because digestion of PCR products can be inefficient, this protocol utilizes primers with sequences that extend beyond the restriction sites and employs overnight digestions with purified DNA. | |
| Procedure |
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A. Polymerase Chain Reaction 1. Prepare two 100 μl Reaction Mixtures in 0.5 μl microcentrifuge tubes as follows (also see Hint #1): 50 μl of scFV DNA (1 to 4 μg, prepare in Protocol ID#2199) 33 μl of ddH2O 10 μl of 10X NEB 3 Buffer 1 μl of 100X Acetylated BSA (supplied with NocI from NEB) 3.0 μl of 10 Units/μl NcoI (NEB) 3.0 μl of 10 Units/μl NotI (NEB) 2. Incubate the Reaction Mixture overnight at 37°C (see Hint #2). B. Reaction Product Purification 1. Separate the gene repertoires by electrophoresis through a 1% Agarose Gel (see Protocol ID#1265). 2. Extract the gene repertoires from the gel. Use Protocol ID#122 or Geneclean™ (Bio 101, Inc also see Hint #3). 3. Resuspend the gene repertoire in 30 μl of ddH2O. 4. Determine DNA concentration by analyzing the gene repertoires on a 1% Agarose Gel with DNA markers of known size and concentration. C. pHEN Preparation for DNA Insert 1. Prepare cesium chloride-purified pHEN 1 DNA (see Protocol ID#571). 2. Digest approximately 4 μg of purified pHEN 1 in a reaction mixture as follows (also see Hint #4): 50 μl of purified pHEN 1 (1 to 4 μg) 33 μl of ddH2O 10 μl of 10X NEB 3 Buffer 1 μl of 100X Acetylated BSA (supplied with NcoI from NEB) 3.0 μl of 10 Units/μl NcoI (NEB) 3.0 μl of 10 Units/μl NotI (NEB) 3. Incubate the Reaction Mixture overnight at 37°C (see Hint #2). D. pHEN 1 Product Purification 1. Separate the pHEN 1 by electrophoresis through a 0.8% Agarose Gel (see Protocol ID#1265). 2. Extract pHEN 1 from the gel. Use Protocol ID#122 or Geneclean™ (Bio 101, Inc also see Hint #3). 3. Resuspend the gene repertoire products in 30 μl of ddH2O. 4. Determine DNA concentration by analyzing the gene repertoires on a 1% Agarose Gel with DNA markers of known size and concentration. |
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| Solutions |
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| This bioProtocol does not use any solutions | |
| BioReagents and Chemicals |
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DNA Restriction Enzyme, NotI NEB 3 Buffer Mineral Oil DNA Restriction Enzyme, NcoI |
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| Protocol Hints |
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1. One microcentrifuge tube for the VH-Vκ scFv repertoire and one microcentrifuge for the VH-Vλ scFv repertoire. 2. For the overnight incubation, use a 37°C incubator or heat block with a heated lid to prevent evaporation, or add a layer of mineral oil as used for PCR reactions (Sigma). 3. The contributor of this protocol suggests using the Geneclean™ Kit. Follow the manufacturer's instructions to isolate the DNA. If using Protocol ID#122, substitute ddH2O for TE Buffer. 4. Efficient digestion of pHEN 1 is very important since a small amount of undigested vector leads to a very large background of non-recombinant clones. Using vector DNA prepared by techniques other than cesium chloride will give significantly lower transformation efficiencies! |
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| Citation and/or Web Resources |
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1. Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001) in press. |
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