Protocols Lunch Box

Preparation of microarrays for SNP mapping

2008-11-24T03:20:00.000-08:00

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

2008-11-24T03:12:00.000-08:00

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

2008-11-24T02:59:00.000-08:00

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)

2008-11-19T01:18:00.000-08:00

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)

2008-11-19T01:05:00.000-08:00

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

2008-11-19T00:56:00.000-08:00

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)

Protocol for positive control sample for 2d-dige

2008-11-18T02:21:00.000-08:00

Box: Protocol for positive control sample for 2d-dige

Taken from: Application of highly sensitive fluorescent dyes (CyDye DIGE Fluor saturation dyes) to laser microdissection and two-dimensional difference gel electrophoresis (2D-DIGE) for cancer proteomics

Published in: Nature Protocols

DIGE stands for difference gel electrophoresis.

Typical 2D images of the proteins extracted from a 1 mm2 area of (a) liver cancer tissue and (b) lung adenocarcinona tissue.

Freezing and shipping skin-derived precursors (SKPs)

2008-04-21T09:38:00.000-07:00

Box: Freezing and shipping skin-derived precursors (SKPs)

Taken from: Isolation of skin-derived precursors (SKPs) and differentiation and enrichment of their Schwann cell progeny

Published in: Nature Protocols

Regeneration test of three rice varieties

2008-04-18T04:01:00.000-07:00

Image Title: Regeneration test of three rice varieties. (a) A japonica variety, "Nipponbare," that has a high-regeneration frequency. (b) A japonica variety, "Koshihikari," that has a low-regeneration frequency. (c) An indica variety, "Kasalath," that exhibits a high-regeneration frequency.

Taken from: A protocol for Agrobacterium-mediated transformation in rice

Transformation of Agrobacterium

2008-04-13T09:07:00.000-07:00

Box: Transformation of Agrobacterium

From: Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants

Published in: Nature Protocols

YEB medium Yeast extract (1.0 g l^-1), beef extract (5.0 g l^-1), peptone (5.0 g l^-1), sucrose (5.0 g l^-1), MgSO₄7H₂O (0.5 g l^-1), pH 7.0. Also include the proper antibiotic resistance selection drug for the FP fusion construct carried by these bacteria.

The Boxed protocol is for a reagent used in the Nature Protocol. It is based on the method described in Storage of competent cells for Agrobacterium transformation.

Quantifying pollen viability in Arabidopsis

2008-04-10T07:50:00.000-07:00

Box: Quantifying pollen viability

From: Fluorescent Arabidopsis tetrads: a visual assay for quickly developing large crossover and crossover interference data sets

Published in: Nature Protocols

The PGM solution used in the Boxed protocol is prepared as follows:
To 950 ml H₂O, add 170 g sucrose (17% wt/vol), 2 ml 1 M CaCl₂ (2 mM), 3.25 ml 0.5 M boric acid (1.625 mM) and 100 mul Triton-X (0.1% vol/vol).

Deriving Linkage Distance and Gene Order From Three-Point Crosses contains some background information about the ABC genotype reference...

BIO-RAD protein assay

2008-04-08T05:07:00.000-07:00

Box: BIO-RAD protein assay

Taken From: Southwestern blotting in investigating transcriptional regulation

Published in: Nature Protocols

This is a short protocol for determining protein concentrations.
The main protocol is open access during 2008 being part of the sample issue.

Preparation of the per-O-trimethylsilylated glucoside 1a and 1b

2008-04-07T09:55:00.000-07:00

Box: Preparation of the per-O-trimethylsilylated glucoside 1a and 1b

Taken from: Regioselective one-pot protection of glucose

Published in: Nature Protocols

Glucosides 1a and 1b, shown in Figure 1 of the protocol, are the starting materials for a series of reactions to protect different combinations of hydroxyl groups in the glucose molecule.

Tissue preparation, microscopy methods and imaging techniques

2008-04-06T06:41:00.000-07:00

Box: Tissue preparation, microscopy methods and imaging techniques

Taken from: Rayburst sampling, an algorithm for automated three-dimensional shape analysis from laser scanning microscopy images

Published in: Nature Protocols

The correct links for the figures are:
Figure 4
Figure 5

Removal of internal peptides by biotinylation and streptavidin purification

2008-04-05T10:09:00.000-07:00

Box: Removal of internal peptides by biotinylation and streptavidin purification

Taken from: Positional proteomics: preparation of amino-terminal peptides as a strategy for proteome simplification and characterization

Published in: Nature Protocols

The boxed method was originally published in Nature Methods in an article entitled "Positional proteomics: selective recovery and analysis of N-terminal proteolytic peptides".

One of the problems with looking at all of the proteins in a cell is that there are a lot of them! A further complication is that most methods of identifying the proteins by mass spectroscopy involve chopping them up into smaller pieces before HPLC separation. This protocol is a way to simplify this mixture by selectively isolating the "pieces" corresponding to the N-termini.

Confirmation of the Mutation by RFLP Analysis

2008-04-05T10:04:00.000-07:00

Box: Confirmation of the Mutation by RFLP Analysis

Taken from: Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease

Published in: Nature Protocols

RFLP Analysis stands for: Restriction fragment length polymorphism analysis.

Preparation of bovine thymocytes

2008-04-05T09:53:00.000-07:00

Box: Preparation of bovine thymocytes

Taken from: Flow cytometry and FISH to measure the average length of telomeres (flow FISH)

Published in: Nature Protocols

I really like the first step in the boxed method: "Arrange for fresh bovine thymus through a local meat packer. Request the collection of 2–3 small (e.g., cubic inch) pieces in a 1,000 ml container filled with digest solution. Arrange for pickup and process immediately."

FISH is another one of these very clever acronyms that make it so that if you search for them in PubMed or Scopus (or anywhere else for that matter) you are initially puzzled by the presence of large numbers of articles that are completely uninteresting to you. In this case FISH stands for fluorescent in situ hybridization.

[moderately amusing aside: the first search that I EVER did on Medline was for PET (as in positron emission tomography), but of course I got quite a lot more than that...]

Separation of dendritic cells

2008-04-05T09:46:00.000-07:00

Box: Separation of dendritic cells

Taken from: Collection of lymph-borne dendritic cells in the rat

Published in: Nature Protocols

The method described in box would be performed after you had collected lymph from the rat (the procedure of the main protocol).

Examples of DNA isolation protocols

2008-04-05T09:35:00.000-07:00

Box: Examples of DNA isolation protocols

Taken from: DNA methylation: Bisulphite modification and analysis

Published in: Nature Protocols

The abovementioned box contains four short protocols for isolating DNA from (1) tissue samples and cell lines, (2) Paraffin block samples, (3) Slide samples, and (4) small numbers of cultured cells (10³-10⁵) after isolation of RNA using TRIzol.

You might used your isolated DNA for any number of things, but in this protocol the idea is to find sites of DNA methylation.

Quote from the abstract: "The process of bisulphite treatment exploits the different sensitivities of cytosine and 5-methylcytosine (5-MeC) to deamination by bisulphite under acidic conditions—in which cytosine undergoes conversion to uracil, whereas 5-MeC remains unreactive."

Preparation of celery juice extract

2008-04-05T08:48:00.000-07:00

Box: Preparation of celery juice extract

Taken from: A protocol for TILLING and Ecotilling in plants and animals

Published in: Nature Protocols

TILLING and Ecotilling sound very agricultural, but (possibly disappointingly) TILLING actually stands for "Targeting-Induced Local Lesions IN Genomes".
So why would you (ever) want to make a celery juice extract? It turns out that celery juice contains an enzyme, a single-strand-specific nuclease, and the extract is used as a reagent in the TILLING protocol.
Here is a picture showing its activity:

Gel image showing the effect of the amount of celery juice extract used to cleave mismatches

This figure was originally published in Nucleic Acids Research.

Introducing Me and My Lunch Box

2008-04-05T08:26:00.000-07:00

Who am I?

Bronwen Dekker, Associate Editor at Nature Protocols and a blogger on the Nature Network.

Why am I creating (yet) another blog?

Well. I wanted a place to try out posting small snippets of content from our site that is available without a site license. Some of it will probably get added to the "Nature Protocols News" type posts in the Nature Network blog, but the people that read that blog might get a bit annoyed if I am posting protocols content every day!