其它PCR方法
· Standard PCR Protocol (Molecular Biology Techniques Manual)
The followings are described in detail
Recommended Reagent Concentrations
Recommended Reaction Conditions
"Hot Start" PCR
Asymmetric PCR for ssDNA Production
Detecting Products
Labeling PCR Products with Digoxigenin
Cleaning PCR Products
Sequencing PCR Products
Cloning PCR Products
PCR Manual (BMB)
This manual contains detailed protocols on performing PCR as well as preparation of templates and post-PCR clean-up. Important applications such as PRINS, cycle sequencing, detection of human chromosomes, and non-radioactive labeling are discussed.
Core Sample PCR
· Core sample PCR (NWFSC)
A method to re-PCR unique bands from products of mixed size
· DNA Amplification in the Presence of Agarose (FMC)
Touch Down PCR
· Touchdown PCR (Alkami Biosystems)
Hot-Start PCR
· "Hot Start" PCR (Molecular Biology Techniques Manual)
Asymmetric PCR
· Asymmetric PCR for ssDNA Production (Molecular Biology Techniques Manual)
Inverse PCR
· Inverse PCR and Cycle Sequencing Protocols (E. Jay Rehm,
Berkeley Drosophila Genome Project)
· Inverse PCR (PMCI)
To Isolate DNA Adjacent To Known Sequence in Genomic DNA
· Inverse PCR (Gottsschling Lab)
Degenerate PCR
· Degenerate PCR Primer Design (Molecular Biology Techniques Manual)
For amplification of cognate sequences from different organisms, or for "evolutionary PCR", one may increase the chances of getting product by designing "degenerate" primers: these would in fact be a set of primers which have a number of options at several positions in the sequence so as to allow annealing to and amplification of a usr/localiety of related sequences.
· Degenerate PCR (Michael Koelle)
· Degenerate PCR (Michael Koelle) (at another server)
Detailed guide in degenerate PCR, including primer design, reaction conditioning and product analysis.
· Degenerate PCR-A Short Guide (Atle M. Bones)
PCR-Elisa
· PCR-Elisa (NUNC)
Run PCR with internal amplification control. After completion of the competitive PCR the target DNA and the control DNA are detected by colorimetric reaction in separate wells on the microtiter plate.
Others
PCR of Blood, Hair or Small Tissue Samples (Gary A. J. Brown)
Amplify DNA from a drop of Blood.
· PCR from Tissue (Schneitz Lab)
Use tissue directly for PCR without DNA extraction
· PCR DNA Biotinylation (KPL)
A rapid method for biotinylating DNA probes through incorporation of biotin-N4-dCTP via a thermostable DNA polymerase in the polymerase chain reaction
· Colony PCR ( Cindy Troxell)
Colony PCR can be used to identify colonies where your favorite gene (yfg) has been replaced with a marker gene by homologous recombination, and to distinguish homologous recombination events from non-homologous. It can also be used to identify colonies from a tetrad that carry a particular gene replacement. This is not a substitute for Southern blotting, but an adjunct.
· Colony PCR (D. Amberg)
It works either for E coli and yeast
· Direct PCR from Whole Yeast Cells (Namjin Chung)
Zymolyase Method for screening yeast transformants
· DIAPOPS Technique (Detection of ImmobilizedAmplified Products) (NUNC)
Introduction to DIAPOPS
· Solid Phase DNA Amplification (NUNC)
· Site-directed Mutagenesis using PCR (NWFSC)
Use of PCR in site-directed mutagenesis accomplishes strand separation by using a denaturing step to separate the complementing strands and allowing efficient polymerization of the PCR primers. PCR site-directed methods thus allow site-specific mutations to be incorporated in virtually any double-stranded plasmid; eliminating the need for M13-based vectors or single-stranded rescue.
· Calculating Concentrations for PCR (Molecular Biology Techniques Manual)
How to calculate primer and nucleotide concentration
· PCR Products Labeling (Amersham)
Guidelines for preparation of radioactively-labeled PCR products for use as probes
· Purification and Sequencing of PCR Product (Donis-Keller lab)
This method is used to sequence directly a specific PCR product after large scale amplification. The reaction should be carried out in the usual fashion, except that after optimization of annealing temperature and other conditions in the 5 µl volume, a large scale 50 µl (10X) total reaction volume should be used to generate sufficient PCR product for sequencing.
· PCR Product Sequencing (Genetics Resources CORE Facility at Johns Hopkins U)
Detailed method for preparing PCR products for direct sequencing
96 Well Plate Purification of PCR products
Purification with Sepahcryl S-300 or S-500 - in filter bottom plates 96-well silent screen plate, Purification with Guanidine-HCl in MES buffer - in glass filter plates
Purification with Guanidine-HCl in KAc buffer
Protocol for PCR of M13
To generate second strands for reverse sequencing of M13 templates
Gene Disruption by Fusion PCR (E. Beasley and D. Amberg)
PCR amplification of rRNA gene fragment. (Jeffrey D. Newman)
PCR Based Gene Deletion (Linda Riles)
The selectable marker for deletion (HIS3) is amplified by PCR using oligos that contain homology to the HIS3 gene and also 45 bp of homology immediately 5' and 3' to the gene being deleted. The PCR product is used to transform yeast to His+, which removes the gene from START to STOP. Correct gene deletion is verified by PCR of yeast colonies, using a universal test oligo in HIS3, and an oligo in flanking chromosomal sequence. Only correctly deleted transformants yield a PCR product.
· Single Tube Confirmation PCR Protocol (SaccharomycesGenome Deletion Project)
A colony PCR strategy is used to identify successful deletions. This method is fast, reliable, and amenable to a 96-well format
· PCR in a teacup (ktotah@sidwell.edu)
PCR in teacup can simply be defined as PCR without a PCR machine. This "old-fashioned" method uses hot plates and water baths to go through the amplification process. Because of its tediousness it is not the most successful way to perform a polymerase chain reaction, but its advantages are that it is inexpensive and simple.
· Non-organic Isolation of Tail DNA and PCR (Jackson Labs)
Using tail tissue directly for PCR after proteinase K digestion.
· Extraction of DNA from Blood for PCR (Jackson Labs)
Simple method for preparing DNA from blood for PCR without organic extraction
· attB Adapter PCR Protocol (LTI)
The Adapter PCR protocol allows for shorter primers to amplify attB-PCR products by utilizing four primers instead of the usual two in a PCR reaction. This protocol is particularly useful when cloning large numbers of genes (universal attB adapter primers can be synthesized once and used hundreds of time), or when additional sequences are added to the template specific primer, such as a TEV protease cleavage site, which result in a long primer.
· PCR for Radiation Hybrid Mapping (Stanford Human Genome Center)
A protocol used to assay for the presence of an STS for radiation hybrid mapping
