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3'-End cDNA Amplification Using Classic RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4130

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO). Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1). - [Read 3'-End cDNA Amplification Using Classic RACE Protocol]

5'-End cDNA Amplification Using Classic RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4131

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT). Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP. Amplification is carried out using three primers. - [Read 5'-End cDNA Amplification Using Classic RACE Protocol]

Additional slide pre-treatment for mapping smaller insert clones - http://www.sanger.ac.uk/HGP/methods/cytogenetics/additional_treatment.shtml

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Additional slide pre-treatment for mapping smaller insert clones for FISH. Sanger Institute. - [Read Additional slide pre-treatment for mapping smaller insert clones]

Agar Plates for Selection of Clones in Bacteria - http://www.le.ac.uk/biology/phh4/bactplte.htm

Category: Microbiology: Bacterial E.Coli Culture Media & Plates

LB Agar: Luria Broth, X-Gal preparation, IPTG and ampicillin. Petri dish preparation. Pat Heslop-Harrison and Trude Schwarzacher, Univ. Leicester - [Read Agar Plates for Selection of Clones in Bacteria]

Amplification of Insert End Sequences from BACs Clones by Thermal Asymmetric Interlaced PCR - http://pubs.nrc-cnrc.gc.ca/ispmb/ispmb16/16175-2.pdf

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol presents the amplification of insert end sequences from bacterial artificial chromosome clones using TAIL-PCR. The amplified products are suitable as probes for chromosome walking and genome mapping and as templates for direct sequencing. The protocol has been used in rice genome studies. - [Read Amplification of Insert End Sequences from BACs Clones by Thermal Asymmetric Interlaced PCR]

Analysis of Clones for Exon Trapping and Amplification - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3651

Category: DNA Protocols: Genomic DNA Library Protocols

Amplification and sequencing of the exon-trapped products. - [Read Analysis of Clones for Exon Trapping and Amplification]

Analysis of ES Cell Clones by Mini-Southern - http://www.imgen.bcm.tmc.edu/molgen/labs/bradley/mini-sou.txt

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Analysis of ES Cell Clones by Mini-Southern. Bradley's Lab, Texas. - [Read Analysis of ES Cell Clones by Mini-Southern]

Analysis of Recombinant Bacteriophage M13 Clones Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3996

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A rapid method to analyze the size of the single-stranded DNA of M13 recombinants. - [Read Analysis of Recombinant Bacteriophage M13 Clones Protocol]

BAC-FISH Protocol - http://www.nhm.ac.uk/hosted_sites/schisto/protocols/BAC_FISH.html

Category: BACs Bacterial Artificial Chromosome Protocols

Protocol uses the BIOPRIME reaction kit from GibcoBRL to prepare biotin-labelled BAC DNA which is detected using FITC-Avidin (Vector Labs, DCS grade). Reagents from other manufacturers may work equally well but have not been tested. Includes: Labeling of BAC clones; Ethanol precipitation; Hybridization; Post-hybridisation treatment / detection. - [Read BAC-FISH Protocol]

Calcium phosphate Tranfection for MDCK cells - http://www.its.caltech.edu/%7Ebjorker/Ca-P_Transfection_MDCK.pdf

Category: Cell Biology and Cell Culture: Cell Transfection Protocol

Protocol for cell transfection, clone screening and picking cell clones. Bjorkman Group. California I.T - [Read Calcium phosphate Tranfection for MDCK cells]

Direct Adaptation of Insect Cells to HyQ© SFX-Insect™ Medium - http://www.hyclone.com/pdf/procedure_insect_directadaptation.pdf

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Using this method, insect cells should be adapted to HyQ SFX-Insect in 4-8 passages. However, there are some cell lines and clones that are more sensitive to the physiochemical and nutritional changes, and in some cases it may take longer than 8 passages for the adaptation. - [Read Direct Adaptation of Insect Cells to HyQ© SFX-Insect™ Medium]

Direct Selection of cDNAs with Large Genomic DNA Clones Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4077

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The goal of this method is to identify transcriptionally active genes in cloned segments of genomic DNA. The protocol uses hybridization and affinity purification to recover biotin-labeled cDNAs that bind to a 500-kb segment of human DNA cloned in a BAC vector. However, the method can be easily adapted to other clones of genomic DNAs cloned in high-capacity vectors. - [Read Direct Selection of cDNAs with Large Genomic DNA Clones Protocol]

DNA Isolation From BAC & PAC Clones Protocol - http://bacpac.chori.org/bacpacmini.htm

Category: BACs Bacterial Artificial Chromosome Protocols

Rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen-Tip method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary. - [Read DNA Isolation From BAC & PAC Clones Protocol]

DNA Isolation From BAC & PAC Clones Protocol - http://bacpac.chori.org/bacpacmini.htm

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols

This is a rapid alkaline lysis miniprep method for isolating DNA from large PAC clones. It is a modification of a standard Qiagen-Tip method that uses no organic extractions or columns. The method works very well for doing analytical restriction digests of PAC clones and can be scaled up if necessary. - [Read DNA Isolation From BAC & PAC Clones Protocol]

dsRNA Synthesis Protocol - http://flyrnai.org/RNAi_FAQ_dsrna_syn.html

Category: siRNA and RNAi Protocols

dsRNA From Clones, Primer Designed dsRNA. Drosophila RNAi Screening Center at Harvard Medical School - [Read dsRNA Synthesis Protocol]

Embryonic Stem Cell growth media Requirements - Taconic Transgenics - http://www.taconic.com/emerging/ESCells/Growth_Media_solutions.htm

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture: ES Cell Growth Media and Requirements

Reference: Michael P. Matise, Wotjek Auerbach and Alexandra L. Joyner (2000). Gene targeting: a practical approach. Protocol excerpted from Chapter 3, Production of targeted embryonic stem cell clones. Alexandra L. Joyner (ed.), 2nd edition, Oxford Unive - [Read Embryonic Stem Cell growth media Requirements - Taconic Transgenics]

Expansion of Targeted ES Clones Protocol - http://escore.im.wustl.edu/htmldocs/xpandtes1.htm

Category: Stem Cell Protocols: Stem Cell Culture Protocols

Protocol for the expansion of targeted ES clones. - [Read Expansion of Targeted ES Clones Protocol]

Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates - http://www.imgen.bcm.tmc.edu/molgen/labs/bradley/freezing.txt

Category: Cell Biology and Cell Culture: ES Stem Cell Biology and Culture

Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates. Bradley Lab Texas. - [Read Freezing Embryonic Stem (ES) Cell Clones in 96-Well Plates]

Generation of a Library of Randomly Overlapping DNA Inserts Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4081

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

Shotgun sequencing of a large segment of DNA involves random fragmentation of the target region into smaller segments that are subsequently cloned into a bacteriophage M13 vector. The goal is to create a library of overlapping clones that provide at least fivefold coverage over the entire length of the target fragment. - [Read Generation of a Library of Randomly Overlapping DNA Inserts Protocol]

Identification of End Clones in YAC Subclone Libraries Protocol - http://humgen.wustl.edu/hdk_lab_manual/yeast/yeast10.html

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

To identify the YAC subclones containing both a human insert and a portion of either the left or right arm of the pYAC4 vector. Identification of these clones is necessary in order to do YAC chromosome walking, and is also useful in the determination of whether a particular YAC clone has a contiguous human insert or whether a co-cloning event has occurred. Vector arm sequences are identified using pBR322 fragments from a BamHI-PvuII double digest. - [Read Identification of End Clones in YAC Subclone Libraries Protocol]

Identification of Positive GATEWAY Expression Clones Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.prot4647

Category: Bacteria Genetics Protocols

Protocol for the identification of positive GATEWAY expression clones when both the pENTRY and pDEST vectors contain the same marker for bacterial selection. Protocol describes ways in which difficult vector combinations can be used effectively to obtain the appropriate expression clone without having to convert the pENTRY clone or pDEST clone to vectors with compatible antibiotic resistances. - [Read Identification of Positive GATEWAY Expression Clones Protocol]

Liquid Phage Lysates Protocol - http://humgen.wustl.edu/hdk_lab_manual/lambda/lambda2.html

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

5 ml liquid lysates are prepared when a small amount of DNA from a large number of lambda clones is needed. The lysates can be made using 10- 20 ul of a stock lysate or a 100-fold amplified phage "macroplaque" as the inoculum. - [Read Liquid Phage Lysates Protocol]

Long Term Lambda Phage Storage Protocol - http://humgen.wustl.edu/hdk_lab_manual/lambda/lambda6.html

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

No special treatment is required to prepare a lysate for the active collection. The following procedure should be used for long-term storage of lambda clones in the archival collections. The phage are diluted in media containing 7% DMSO and frozen at -80 degrees C. - [Read Long Term Lambda Phage Storage Protocol]

Microinjection of Mouse ES Cells into Blastocysts - http://www.bcm.edu/dtmc/?pmid=7791

Category: Microinjection Protocols: Microinjection of DNA Protocols

The core defines one injection as a total of forty blastocysts injected on two consecutive days using one or two clones for the same mutation. Please, refer to the Pricing page for information on the cost of one injection. - [Read Microinjection of Mouse ES Cells into Blastocysts]

Monoclonal Antibody Production - http://research.cm.utexas.edu/bkitto/Kittolabpage/Protocols/Immunology/MAb.html

Category: Antibody Protocols: Antibody Production Protocols

Monoclonal Antibody Production. Includes: Mouse Immunization; Bleeding Mice; Hybridoma Fusion; Assaying for Positive Clones; Expanding Positive Clones. - [Read Monoclonal Antibody Production]

Articles (1-3 of 3)

Picking Transfected ES Cell Clones Protocol

This protocol a protocol on how to generate transfected embryonic stem (ES) cell clones. The previous protocol in this series is the Protocol for Electroporation of ES cells. The next protocol in the series is the Protocol on Disaggregation, Expansion, and Freezing of Transfected ES Clones.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

Vector Design Protocol for Transgenic Mice Generation

The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.

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