Monday, 24 November 2008

Preparation of microarrays for SNP mapping

Box: Preparation of microarrays

Taken from: Positional cloning by fast-track SNP-mapping in Drosophila melanogaster

Published in: Nature Protocols

TAMS assay

(a) Components used for the hybridization of TAMS products. ... (b) An 'array-of-arrays' format is used to analyze multiple SNPs in many samples in parallel. The two different configurations of the system make it flexible with the possibility to easily change the numbers of SNPs and samples to be interrogated (196 SNPs in 80 samples or 576 SNPs in 96 samples). The dimensions for both formats used for array spotting are indicated, e.g., the diameters of the spots and the silicon grid chambers as well as the center-to-center distances between two spots and two subarrays. The spot measures may vary slightly depending on the needle used for spotting. (c) Four color mini-sequencing in solution in which primers hybridize next to each SNP site and are extended with fluorescently labeled terminating nucleotide analogs (ddNTPs). ... (d) On the left is an example of one subarray scanned at the wavelength visualizing incorporated ddCTP nucleotides labeled with TAMRA fluorophore. Enlargement of the mapping result for C233, an eyes absent allele, is shown on the right. The entire mapping result is depicted in Figure 5. Black bars indicate the determined genetic interval from the stage 1 mapping.

Protocol for preparation of electrocompetent JUDE-1 E. coli cells carrying plasmid pBAD33-NlpA-ZZ

Box: Protocol for preparation of electrocompetent JUDE-1 E. coli cells carrying plasmid pBAD33-NlpA-ZZ

Taken from: E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display

Published in: Nature Protocols

Assay of Phospholipid Concentration by the method of Bartlett

Box: Assay of Phospholipid Concentration by the method of Bartlett

Taken from: Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+–ATPase as an example

Published in: Nature Protocols

This method can be used to determine the total phospholipid concentration in a solution. Provided of courise that you aren't using a phosphate buffer!

Reference 46 = Bartlett, G.R. Phosphorus assay in column chromatography. J. Biol. Chem. 234, 466–468 (1959)

Wednesday, 19 November 2008

Box: Slide pretreatment with pepsin to remove cytoplasm

Taken from: Spectral karyotyping analysis of human and mouse chromosomes

Published in: Nature Protocols

"Check the slide under a light microscope using a times40 high-dry phase objective. If cytoplasm is present (gray particulate matter surrounding the chromosomes; see Fig. 2a), pretreat the slides with pepsin as described in Box 1. If cytoplasm is not visible (a clear light margin surrounds the metaphase chromosomes) and the chromosomes have good morphology (black in color, not phase-light or shiny), there is no need for slide pretreatment with pepsin." (Quoted from the protocol)

Effects of humidity and removal of excess cytoplasm by slide pretreatment on hybridization efficiency.

(a) When cells are not kept in the hypotonic solution for enough time, the chromosomes become trapped inside the cytoplasm (white arrows). Altering the humidity at which the metaphase preparation is dropped onto the microscope slide may sometimes alleviate this problem; otherwise, hybridization efficiency will be compromised. (b) When normal human metaphase spreads are pretreated with pepsin and hybridized with a whole chromosome paint probe labeled with tetramethyl rhodamine isothyocyanate (TRITC), the hybridization will be successful. (c) Here, a whole chromosome paint-labeled with TRITC was hybridized to a different normal human metaphase spread and the slide was not pretreated with pepsin. Note the weaker intensity of the hybridization signal (red color).

Acquisition and maintenance of BY-2 cells (tobacco plant cells)

Box: Acquisition and maintenance of BY-2 cells

Taken from: Cell cycle synchronization of tobacco BY-2 cells

Published in: Nature Protocols

RetroNectin treatment for colocalization of virus and target cells

Box: RetroNectin treatment for colocalization of virus and target cells

Taken from: Use of a lentivirus/VSV pseudotype virus for highly efficient genetic redirection of human peripheral blood lymphocytes

Published in: Nature Protocols

"RetroNectin may be used to facilitate colocalization of virus and target cells. This is particularly important for retrovirus transduction but can generally be omitted when using Lentivirus/VSV pseudotyped virus. It is included here should readers elect to use retroviruses for rapidly dividing cell targets." (Quoted from the box)