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Nco I and Not I Restriction Digestion of the scFv Gene Repertoires
Contributor:
The Laboratories of Andrew Bradbury at Los Alamos National Laboratory and James D. Marks University of California, San Francisco
Overview
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
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.
Solutions
This bioProtocol does not use any solutions
BioReagents and Chemicals
DNA Restriction Enzyme, NotI
NEB 3 Buffer
Mineral Oil
DNA Restriction Enzyme, NcoI
Protocol Hints
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!
Citation and/or Web Resources
1. Phage Display: A Practical Approach. Eds. T. Clackson and H. Lowman. Oxford University Press, (2001) in press.
