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Peer-Reviewed Protocols
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Wai Hung Tsang and King L. Chow Peer-reviewed, author-submitted protocol. Published in November 2009
pp55- 56
Procedure 1. A pair of fine forceps with a region of ~1 mm in width is used to hold the thin end of an autoclaved gel-loading tip in a perpendicular position (Figure 1).
Kim M. Linton1,5*, Yvonne Hey2*, Sian Dibben2, Crispin J. Miller3, Anthony J. Freemont4, John A. Radford1,5, and Stuart D. Pepper2 Peer-reviewed, author-submitted protocol. Published in November 2009
Protocol overview • This protocol covers tissue sectioning and macrodissection steps in preparation for RNA extraction from FFPE tissues. Although originally developed for RNA extraction using the Optimum FFPE RNA Isolation Kit (Ambion, UK), the protocol is relevant for FFPE tissue preparation for RNA extraction using any extraction method • Macrodissection is carried out at 2× magnification using a dissecting microscope with external fiber-optic illumination • Adopt strict RNase-free handling conditions throughout, including the use and frequent changing of powder-free latex gloves.
Kim M. Linton1,5*, Yvonne Hey2*, Sian Dibben2, Crispin J. Miller3, Anthony J. Freemont4, John A. Radford1,5, and Stuart D. Pepper2 Peer-reviewed, author-submitted protocol. Published in November 2009
Protocol overview • The extraction method (steps 2–21) is taken from the method supplied with TRIzol reagent (Invitrogen, Paisley, UK). • Recover tumor tissue at the time of surgery, trim into 1-cm3 fragments, and immerse immediately in TRIzol reagent prior to freezing at -80°C.
Michael A. Green1, Shannon Bass2, and Brett T. Spear1,3 Peer-reviewed, author-submitted protocol. Published in November 2009
Instructions for use For the production of zygotes and pseudopregnant females to serve as recipients, standard transgenic methodologies are used. Matings are set up with male and female mice as in standard transgenic procedures.
Yong-Chul Jung1, Jia Xu2, Jun Chen1, Yeong C. Kim1, David J. Winchester3, and San Ming Wang1 Peer-reviewed, author-submitted protocol. Published in November 2009
pp46- 47
Depending on the desired resolution and sequencing scale, different restriction enzymes can be used for genomic DNA digestion. PstI is used here as an example.
Hélène Labit1, Arach Goldar2, Guillaume Guilbaud1, Carine Douarche3,4,2, Olivier Hyrien1, and Kathrin Marheineke1 Peer-reviewed, author-submitted protocol. Published in November 2009
pp48- 50
Procedure 1. Loosely place 25 coverslips (22 × 22 mm) into a clean 250 mL beaker.
Peer-reviewed, author-submitted protocol. Published in BioTechniques Protocol Guide 2009
BL21-Gold(DE3) Cells (Stratagene, La Jolla, CA, USA) Glycerol (Merck, Nottingham, UK) IPTG (Sigma, Madrid, Spain) Arabinose (Sigma) Nikon Eclipse-600 Microscope equipped with a 100× oil immersion objective, a filter set for GFP (Nikon, Badhoevedorp, The Netherlands) Nikon DS-5Mc Cooled Camera (Nikon) Complete Protease Inhibitor Cocktail (Roche, Barcelona, Spain) Lysozyme (Sigma) Bio-Rad IQ5 System (Bio-Rad Laboratories, Madrid, Spain) β-Mercaptoethanol (Sigma) DDT (Sigma) 2× Laemmli Sample Buffer (Invitrogen, Barcelona, Spain) Ni-Sepharose 6 Fast Flow (Amersham Biosciences, Barcelona, Spain) Vector Plasmids Expression vectors encoding the N- and C-terminal segments of the GFP mutant sg100 fused to two leucine zipper-coding sequences (designed as NGFP-LZ1 and LZ2-CGFP) and two control vectors (designated as CGFP and NGFP without LZ1 or LZ2 sequences) were a generous gift of Thomas J. Magliery (1, 2).
Vendor-submitted protocol. Published in November 2009
ReagentsSource of Genomic DNA (e.g., liver)Tris Base (Cat# T1503-1KG; Sigma-Aldrich, Gillingham, UK)Ethylenediaminetetraacetic Acid (EDTA; Cat# 100935V; VWR, Lutterworth, UK)Sodium Dodecyl Sulphate (SDS; Cat# L-4509; Sigma-Aldrich)Proteinase K (Cat# P2308-25MG; Sigma-Aldrich)Ethanol [70%; take 70 ml absolute ethanol (Cat# 1010774Y; VWR) and add 30 ml of sterile water]Acetone (Cat# 100035R; VWR)N,N-Dimethylformamide (DMF; Cat# D4551-500ML; Sigma-Aldrich)KOD Hot Start DNA Polymerase Kit, with 200..
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