The pattern and extent of DNA methylation can significantly affect the success of restriction digestions
or bacterial transformations. Prokaryotic DNA may be methylated by host restriction/modification
systems, while eukaryotic DNA often is methylated i - [Read A practical guide to DNA methylation Promega PDF]
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]
4 strains of E. coli are used in these studies: JM101 for M13 infection and isolation, XL1BMRF'for M13 or pUC-based DNA transformation, and ED8767 for cosmid DNA transformation. To maintain their respective F' episomes necessary for M13 viral infection, JM101 is streaked onto a M9 minimal media plate and XL1BMRF' is streaked onto an LB plate containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid.. - [Read Bacterial Cell Maintenance Protocol]
To 2mls of mid-log culture or 1ml of freshly saturated culture add 1ml(or an equal volume) of glycerol solution, mix gently, then freeze rapidly in liquid nitrogen or on dry ice. Store at -20°C and - 70°C. - [Read Bacterial Glycerol Stocks Protocol]
Protocol for C. elegans RNAi. Includes: Transformation of competent cells; Blunt-end ligation; Preparation of competent cells; Dephosphorylation of linear plasmid DNA; Restriction Digest: EcoRV; Insert amplification from gDNA; Gel purification: QiaQuick gel purification kit; Mini-prep; Transformant Screening; Bacterial preparation and induction; Preparation of worms for RNAi feeding. - [Read C. elegans RNAi Protocol]
Nice protocol for the preparation of Bacterial cells to allow uptake of plasmid vectors. Dr Peter Kille. Hot Metals Cardiff. - [Read Competent Cell Preparation [CaCl2]]
Direct sequencing from amplified bacterial and large insert cloned human or mouse genomic DNA via an improved MultiPlex PCR-based method. Includes: Preparing primers for MP-PCR; Amplification; PCR Product Clean-up; Sequencing. - [Read Direct Sequencing Using MultiPlex PCR-Based Method]
Information on the diseases of Xenopus tropicalis. Includes: PREMETAMORPHS: Bacterial Infection of Embryos; Protozoal Infestation of Tadpoles; POSTMETAMORPHS: Mycobacteriosis; Chytridiomycosis (Batrachochytrium dendrobatidis). - [Read Diseases of Xenopus tropicalis]
Protocol describes a method for electroporating DNA into ES cells, as well as selection methods. Pilot studies should be performed to optimize the conditions for each DNA construct. The selection method described here is one of the most complex. It involves targeting constructs in which the bacterial neomycin-resistance gene disrupts the coding sequence of the mouse gene. - [Read Electroporating DNA into Embryonic Stem (ES) Cells and Selection Methods Protocol]
Fixation is to preserve cells and tissue constituents in as close a life-like state as possible and to allow them to undergo further preparative procedures without change. Fixation arrests autolysis and bacterial decomposition and stabilises the cellular and tissue constituents so that they withstand the subsequent stages of tissue processing. Great detailed guide with protocols. Anthony S-Y Leong. - [Read Fixation and Fixatives - A great guide]
A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.