PCR引物设计

Methylated CpG Island AmplificationProtocol written by Minoru Toyota2. Materials2.1. MCARestriction enzymes SmaI XmaI T4 DNA ligase Taq DNA polymerase 10X PCR reaction buffer: 670mM Tris-HCl pH 8.8 40 ...

Primer Design Rules for Bisulfite Conversion Based PCRAuthor: Long-Cheng LiSource: Protocol OnlineAbstract: Primer design rules for MSP and bisulfite sequencing PCR. A. General consideration for pri ...

Methylation-Specific PCRProtocol written by James HermaundefinedMethylation-specific PCR (MSP) is a simple rapid and inexpensive method to determine the methylation status of CpG islands. This approach allo ...

Singel Nucleotide Primer Extension (SNuPE)Contributed by Dr. A. GratchevSingle Nucleotide Primer Extension is a powerful method which can be used for the precise analysis of methylation in a certain p ...

Bisulfite-PCR for Restriction Analysis and/or SequencingProtocol written by Jean-Pierre Issa based on several published papers.Bisulfite-PCR followed by restriction is a rapid and semi-quantitative me ...

TABLE OF CONTENTS 1. Abstract 2. Introductory statement 3. The key preparatory steps 3.1. Fixative 3.1.1. Protease digestion 3.1.2. Definition of optimal protease digestion 3.1.3. Definitions of subop ...

Inverse PCR (IPCR) described by Ochman et al in 1988 is a method for the rapid in vitro amplification of DNA sequences that flank a region of known sequence. The method uses the polymerase chain react ...

Inverse PCR for PAC-end sequencing from Brad Barbazuk Goal is to generate PCR fragments that contain the ends of PAC inserts that can be sequenced. For inverse PCR we cut the PAC once in the vector ( ...

IntroductionThe diversity of pathogenesis presents a spectrum of challenges to the researcher who is required to utilize a wide variety of tools and techniques including those of a histological immun ...

Inverse PCRI. Genomic DNA Prep• from 5 ml culture resuspend in 50 µl TEII. Digestions genomic DNA 5 µl 10x of appropriate NEB buffer 5 µl 0.5 µg/µl RNase 1 µl H2O 38 µl restriction enzyme 1 µl • Use ...

I. Quick Fly Genomic DNA Prep Standard fly mini prep (30 flies) resuspended in 150 ul TE 1) Collect 30 anesthetized flies in eppendorf tube and freeze at -80°. 2) Grind flies in 200 µl Buffer A with ...

Multiplex PCR is a variant of PCR which enabling simultaneous amplification of many targets of interest in one reaction by using more than one pair of primers. Since its first description in 1988 by C ...

PCR and multiplex PCR guide Table below lists the parameters influencing the PCR reaction and indicates some PCR and multiplex PCR applications. Clicking on an item in the table will take you to a det ...

Troubleshooting for PCR and multiplex PCRTroubleshooting discussion is based on the PCR protocol as described in the table below. All reactions are run for 30 cycles.COMPONENTVOLUMEFINAL CONCENTRATION ...

Microdeletion screeningOne application of multiplex PCR is microdeletion screening. This can be applied to the X and Y chromosomes (male genomic DNA) or to hybrid cell lines (rodent-human) containing ...

Standard multiplex mixturesOver 75 primer pairs were chosen and a number of multiplex mixtures were designed and used for different purposes. Examples of all multiplex mixes are presented below.(All u ...

End repair: Add 5-10 units of T4 DNApol and incubate at 37C for 5 minutes. Make solution to 2.5 M NH4OAc and add 1 volume of 95% ethanol; let sit for 5 minutes at RT and spin at 14K x g for 20 minutes ...

When designing PCR experiments in which the synthesized DNA fragment is to be subsequently digested with a RE it is very important to determine how many extra nucleotides should be added to the 5’-en ...

PURIFICATION OF PCR PRODUCTS WITH SEPHADEXPlace the sephadex measuring plate (MultiScreen ?Column Loader) on a clean piece of saran wrap. Pour some sephadex onto the plate (takes about 3.3 g). Scrape ...

Protocol for Real-Time RT-PCRThis protocol describes the detailed experimental procedure for real-time RT-PCR using SYBR Green I as mentioned in Xiaowei Wang and Brian Seed (2003) A PCR primer bank fo ...