Protocol Matches: 282
(Long-Cheng Li, UCSF)
A free online program for designing primers for methylation specific PCR (MSP) and bisulfite sequencing PCR.
Added: Thu May 29 2003, Hits: 2355, Reviews: 0
Telomeric Repeat Amplification Protocol (TRAP) Telomerase Assay
(The Science Advisory Board)
This protocol describes a semi-quantitative in vitro assay to detect telomerase activity. Any active telomerase present in a cell extract from detergent-lysed cells will add a variable number of telomeric repeats (TTAGGG) to the 3' end of a substrate oligonucleotide. PCR that incorporates a radiolabeled nucleotide is then used to amplify the extension products. The presence of telomerase in the cell extracts is assessed by the appearance of a ladder of PCR products on a native polyacrylamide gel.
Added: Mon Feb 23 2009, Hits: 603, Reviews: 0
Multiplex amplification of ancient DNA
(Molecular Ecology Group, Max Planck Institute)
This method allows amplification of multiple PCR fragments (in our hands at least about 50) in a single PCR, saving DNA extract and work. We found it to work well for both mitochondrial and nuclear DNA. Ideally, a complete mtDNA genome can that way be amplified in just two PCRs.
Added: Fri Feb 13 2009, Hits: 9615, Reviews: 0
- Protocol for Real-Time RT-PCR (Dr. Xiaowei Wang at the Department of Molecular Biology, Massachusetts General Hospital)
This protocol describes the detailed experimental procedure for real-time RT-PCR using SYBR Green I. The procedure begins with reverse transcription of total RNA. The cDNA is then used as template for real-time PCR with gene specific primers. You may need to modify this protocol if you use different reagents or instruments for real-time PCR.
Added: Fri May 14 2004, Hits: 2091, Reviews: 0
Gene-specific RT-PCR (Cancer Gene Anatomy Project, NCI)
Protocol may be used for amplifying individual transcripts from RNA recovered from microdissected cell populations. The procedure for reverse transcription is as regular RT, but for PCR, radioactive dCTP is used and PCR products are separated by running polyacrylamide gel.
Added: Tue May 14 2002, Hits: 666, Reviews: 0
- Making T-A cloning Vectors (Chin-Sang Lab, Queen's University, Kingston, ON, Canada)
A method for direct cloning a PCR product, by the T-vector technique. This is cheap and easy way to clone PCR products with A 3’ overhangs.
Added: Fri Feb 06 2009, Hits: 4229, Reviews: 0
The following procedures are described:
RNA extraction and qualification DNase treatment of RNA sample
Separation on acrylamide gels
DNA extraction from bands of interest
Re-amplification by PCR
Separation on agarose gels
Excision of amplified products
Added: Tue May 14 2002, Hits: 1522, Reviews: 0
Microarray Development Protocols
(Vodkin Laboratory, University of Illinois)
Protocols used for a whole microarray experiment including: Preparation of DNA for microarrays / Electrophoresis of PCR products /Purification of PCR products with Sephadex / Preparing poly-L-lysine coated slides / Postprint slide treatments / Postprint blocking of amine slides
Postprint blocking of aldehyde slides / Reverse transcriptase labeling with Cy3/Cy5 / Staining of total DNA on slides /Microarray construction /Laser scanning
Statistics of expression
Sample tracking /Controls used in the microarrays
Added: Mon Aug 26 2002, Hits: 1919, Reviews: 0
- Bisulfite Modification (Conversion) of DNA (Protocol Online)
Modifying DNA using sodium bisulfite to convert unmethylated cytosines to uracils and subsequently detect methylated cytosines using methylation specific PCR (MSP) technique or bisulfite genomic sequencing after PCR amplification with or without cloning. This protocol also describes procedure for handling nanogram quantities of DNA.
Added: Fri May 07 2004, Reviews: 0
- Semi -Quantitative RT-PCR (Pamela Stanley Lab Wiki, Yale University)
The RT-PCR method can be used not only to detect specific mRNAs but also to semi-quantitate their levels. Thus, one can compare levels of transcripts in different samples. This can be done in two different ways. One is to quantitate against levels of transcripts from a control, house-keeping gene (such as actin and GAPDH). (Transcription of house-keeping genes is believed to be unaffected by almost all experimental conditions.) The second method is to add an exogenous, primer-specific PCR template during PCR.
Added: Fri Mar 20 2009, Hits: 7014, Reviews: 0
DNA Amplification Fingerprinting Protocol
(C. S. Prakash)
DNA is amplified by PCR. The resulting PCR products are separated on polyacrylamide gel and silver stained for DNA visualization.
Added: Tue May 14 2002, Hits: 242, Reviews: 0
(Dr. Xiaowei Wang at the Department of Molecular Biology, Massachusetts General Hospital)
PrimerBank is a public resource for PCR primers. These primers are designed for gene expression detection or quantification (real-time PCR). There are several ways to search for primers: by GenBank Accession, NCBI protein accession, LocusLink ID, PrimerBank ID or Keyword (gene description). PrimerBank contains about 180,000 primers covering most known human and mouse genes.
Added: Fri May 14 2004, Hits: 4815, Reviews: 0
Primo online is a friendly PCR primer design tool. It reduces PCR noise by lowering the probability of random primering. Batch mode allows designing for multiple sequences. Users can also calculate the melting temperature using the nearest neighbor model.
Added: Tue Oct 08 2002, Hits: 3012, Reviews: 0
Small-scale DNA prep from Neurospora
(Fungal Genetics Stock Center)
Molecular biology experiments often require preparation of small amounts of DNA from many samples. This abbreviated DNA isolation method yields an average of 0.6 micrograms of genomic DNA that is suitable for Southern analysis or PCR
Added: Mon Dec 09 2002, Hits: 1143, Reviews: 0
Preparation of Template RNA for RT-PCR (Roche Diagnostics Corporation)
Guidelines for preparing high quality RNA template for RT-PCR.
Added: Sat Sep 21 2002, Hits: 779, Reviews: 0
In this protocols, the following procedures are introduced:
Bst-catalyzed radiolabeled DNA sequencing
Radiolabeled sequencing gel preparation, loading, and electrophoresis
Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers
Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions
Sequenase[TM] catalyzed sequencing with dye-labeled terminators
Fluorescent-labeled sequencing gel preparation, pre-electrophoresis, sample loading, electrophoresis, data collection, and analysis on the ABI 373A DNA sequencer
Double-stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers
cDNA sequencing based on PCR and random shotgun cloning
Added: Tue May 14 2002, Hits: 1589, Reviews: 0
(Hubbard Center for Genome Studies)
AFLP is a method for genotyping individuals for a large number of loci using a minimal number of PCR reactions.
Added: Wed Feb 11 2009, Hits: 982, Reviews: 0
DNA Isolation from Buccal Cell
(Daniel T. O'Connor, UCSD Chromaffin Cell and Hypertension Research )
Simple method for obtaining DNA from buccal cells for PCR.
Added: Tue Feb 17 2004, Hits: 5416, Reviews: 0
- dsRNA Synthesis Protocol (Drosophila RNAi Screening Center at Harvard Medical School)
Generating dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends.
Added: Wed May 12 2004, Hits: 2178, Reviews: 0
Ligase independent cloning
(Dr. Arie Geerlof)
Ligase independent cloning (LIC) is a simple, fast and relatively cheap method to produce
expression constructs. It makes use of the 3'--> 5'-activity of T4 DNA polymerase to create
very specific 10-15 base single overhangs in the expression vector. PCR products with
complementary overhangs are created by building appropriate extensions into the primers and
treating them with T4 DNA polymerase as well. The annealing of the insert and the vector is
performed in the absence of ligase by simple mixing of the DNA fragments. This process is
very efficient because only the desired products can form.
Added: Sun Oct 25 2009, Hits: 2421, Reviews: 0
- Chromatin Immunoprecipitation from Human Embryonic Stem Cells (Journal of Visualized Experiments)
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays.
Added: Mon Feb 09 2009, Hits: 2798, Reviews: 0
Choice of reverse transcription primers
(Roche Diagnostics Corporation)
Oligo dT or random hexanucleotides primer, which to use?
Added: Sat Sep 21 2002, Hits: 296, Reviews: 0
YPD-filled microtiter plates
(Linda Riles, (Saccharomyces Genome Deletion Project, Stanford)
For growing individual strains to use for PCR
and shipping strains
Added: Tue May 14 2002, Hits: 867, Reviews: 0
Non-Radioactive EMSA Protocol
(Hammer Lab, University of Michigan)
Procedures include probe preparation by PCR amplifying a fragment of the promoter of interest using DIG-labeled dUTP in addition to normal dNTPs, Standard Nuclear Protein Extraction, Protein/Probe Binding, Electrophoresis, transfer and DIG detection
Added: Mon Mar 16 2009, Hits: 4765, Reviews: 0
Some general guidelines on the primer design for PCR cloning
Added: Tue May 14 2002, Hits: 4516, Reviews: 0
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