Lab Protocols

The light microscope, so called because it employs visible light to detect small objects, is probably the most well-known and well-used research tool in biology. Yet, many students and teachers are unaware of the full range of features that are available in light microscopes. Since the cost of an instrument increases with its quality and versatility, the best instruments are, unfortunately, unavailable to most academic programs. However, even the most inexpensive "student" microscopes can provide spectacular views of nature and can enable students to perform some reasonably sophisticated experiments. A beginner tends to think that the challenge of viewing small objects lies in getting enough magnification. In fact, when it comes to looking at living things the biggest challenges are, in order, obtaining...

Considerations for use The principle of the biuret assay is similar to that of the Lowry, however it involves a single incubation of 20 min. There are very few interfering agents (ammonium salts being one such agent), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods. However, the biuret assay consumes much more material. The biuret is a good general protein assay for batches of material for which yield is not a problem. The Bradford assay is faster and more sensitive. Principle Under alkaline conditions substances containing two or more peptide bonds form a purple complex with copper salts in the reagent. Equipment In addition to standard liquid handling supplies a visible light spectrophotometer is needed, with maximu...

Considerations for use The bicinchoninic acid (BCA) assay is available in kit form from Pierce (Rockford, Ill.). This procedure is very applicable to microtiter plate methods. The BCA is used for the same reasons the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replace the Lowry because it requires a single step, and the color reagent is stable under alkaline conditions. Both a standard assay for concentrated proteins and a micro assay for dilute protein solutions are described below. Principle BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. The advantage of BCA is that the reagent is fairly stable under ...

Considerations for use See considerations listed under the absorbance assay at 280 nm. This method is just as convenient as for absorbance at 280 nm. It may be preferred if there is excessive contamination by nucleic acids, since nucleic acids absorb very little radiation at 205 nm. Setting the wavelength is a bit tricky since 205 nm is right on the shoulder of the protein peak. Principle See the discussion for the 280 nm absorbance assay. Equipment In addition to standard liquid handling supplies a spectrophotometer with UV lamp and quartz cuvette are required. Procedure Include 0.01% Brij 35 in the buffer to prevent adsorption of protein onto plastic or glass surfaces. This is necessary for the 205 nm assay because losses are proportionately higher ...

  Atmospheric Deposition Sample Processing Lab prep: 1. Set aside all necessary bottles, vials, tubes, etc. 2. Locate and cache all necessary glassware and filters. 3. Record bottle numbers on the field datasheet, label N tubes and cation bottles if adequate volume is expected. Label sweater tube trays and sweater boxes. Lab cleanup: 1. Rinse, dry and HCl wash a batch of glassware. 2. ...

ADP flameAA cation Method Detection Limit results (SMQ 3/1/01) K: 0.03ppm K trial (10 reps) S.D. 0.003797 (2.821) 0.0107, MDL 5x the MDL @0.05, this is the Practical Quantitation Limit A new standard range of 0.05-0.5 ppm will need to be used Mg: 0.01ppm Mg trial (10 reps) S.D. 0.00135 (2.821) 0.0038, MDL 5x the MDL @0.02, this is the Practical Quantitation Limit The current standard range of 0.02-0.2 ppm will not change Ca: 0.02ppm Ca trial (10 reps) S.D. 0.006596 (2.821) 0.01967, MDL 5x the MDL @ 0.1, this is the Practical Quantitation Limit The current standard range of 0.5-5 pp...

Description From the lab of Lucien T. "Tres" Thompson, Ph.D. The University of Texas at Dallas Protocol for Radial-Arm Maze Training for Unit Recording By Tim Goble Introduction: Radial-arm training will differ for place-cell recording versus other behavioral paradigms. The emphasis for place-cell recording is on fast, reliable recording sessions where the rat transverses to each arm at least once. Other behavioral paradigms may emphasize number of visits, the order of arms transversed, and/or remembered previous sessions. This training protocol is sufficient for place-cell unit recording sessions. You may need to adapt this protocol for things of specific importance in other paradigms. I. Food Depriving Rats Items Needed: Rat Rat Cage Rat Weight Scale ...

Dependable detection of Xylella fastidiosa (Xf) in glassy-winged sharpshooters (GWSS) is imperative for understanding Xf epidemiology and optimizing grapevine protection strategies. In this study, we have developed methods for extracting Xf DNA from GWSS vectors and optimized a SYBR green I based real-time PCR detection protocol that is fast, consistent, and inexpensive. The Qiagen DNeasy Tissue kit (Qiagen Inc., Hercules, CA, USA) was the most efficient kit tested in our studies, having a lower detection limit of 500 cells in the presence of insect tissue. The considerably faster pre-extraction method of repeatedly flushing the foregut with lytic buffer with vacuum pressure prior to extraction using Qiagen DNeasy Tissue kit was not significantly different than whole-tissue maceration. Storage of GWSS samples at -4°C did not compromise Xf-detection capabilities.

Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust ...

Written by David S. Fay, Dan Starr, Andy Spencer, and Wade Johnson Edited by Amy Fluet and John Yochem What this guide is and isn't. This guide is neither a basic text in Mendelian genetics nor is it in any way a comprehensive description of C. elegans biology. Detailed information about the latter can be gleaned from C. elegans I and C. elegans II by Cold Spring Harbor Press Inc. This guide does assume a working knowledge of basic genetics and will be of limited use to those who lack some background in this area. It is our hope that this text may serve as a supplement to existing published materials and that it will facilitate the successful breeding of worms by those new to the field. Source Link : http://www.mcb.ucdavis.edu/faculty-labs/starr/publication%20pdf%20pictures/2001%20WBFSG.pdf

Step 1 Suspend 2.5lb/1,000 gal (30g/hL) of Go-Ferm in 20 times its weight of clean 110°F (43°C) water. IMPORTANT: If not using Go-Ferm, water temperature should be 104ºF (40°C)to avoid damaging the yeast. Step 2 Once the temperature of the GO-FERM solution has dropped to 104°F (40°C), add 2lb/1,000 gal (25g/hL) of active dried yeast. Stir gently to break up any clumps. Let suspension stand for 15-30 minutes, then stir gently again. Note: Foam is not an indicator of yeast viability. STEP 3 Slowly (5 minutes) combine an equal amount of must to be fermented with the yeast suspension. This will help the yeast to adjust to cool temperature must and avoid cold shock caused by a rapid temperature drop exceeding 18°F (10°C). This atemperation may need repeating in very low temperature must. Step 4 Add the yeast slurry to the bottom of the fermentation vessel just as you begin filling the vessel with must. source Link :http://www.lallemandwine.us/pdf/articles/Rehy_eng_14-a...

Acridine orange/ethidium bromide (AO/EB) staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. Cells are viewed under a fluorescence microscope and counted to quantify apoptosis. Shailaja Kasibhatla, Gustavo P. Amarante-Mendes, Deborah Finucane, Thomas Brunner, Ella Bossy-Wetzel and Douglas R. Green Details of this protocol, Acridine Orange/Ethidium Bromide (AO/EB) Staining To Detect Apoptosis (Subscription Required), are located on a web site other than Biocompare Protocols. Source Link : http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4493

Scanning probe microscopy covers several related technologies for imaging and measuring surfaces on a fine scale, down to the level of molecules and groups of atoms. At the other end of the scale, a scan may cover a distance of over 100 micrometers in the x and y directions and 4 micrometers in the z direction. This is an enormous range. It can truly be said that the development of this technology is a major achievement, for it is having profound effects on many areas of science and engineering. SPM technologies share the concept of scanning an extremely sharp tip (3-50 nm radius of curvature) across the object surface. The tip is mounted on a flexible cantilever, allowing the tip to follow the surface profile (see Figure). When the tip moves in proximity to the investigated object, forces of interaction between the tip and the surface influence the movement of the cantilever. These movements are detected by selective sensors. Various interactions can be studied depending on t...

Synthesis of Double-Stranded cDNA from Purified Poly(A) mRNA àUse T7-(dT)24 oligomer for priming first-strand cDNA synthesis: 5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3’ High-quality HPLC-purifed T7-(dT)24 primer should be used (NOT PAGE-purified primer). This is available from GENSET. 1st strand synthesis starting material: 0.2-5mg poly (A) mRNA àdetermine correct volumes of H2O and reverse transcriptase to be used: for every mg of RNA, need 1ml of SSIIRT (200U/ml). For mRNA quantity Procedure: 1. To 1.5mL RNase-free polypropylene tube, add: Volume final concentration 1. T7-(dT)24 primer 1ml 100pmol 2. mRNA 0.2 to 5 mg 3. DEPC-treated H2O enough to bring final volume (after step3) to 20ml 2.Add to tube: a. 5X first strand buffer 4ml 1X b. 0.1M DTT ...