ברצוננו להודות לקרן קנדית עבור חדשנות, פיתוח קולומביה הבריטית קרן ידע מדעי הלאומי וההנדסה מועצת המחקר (NSERC) של קנדה לתמיכה מחקרים מתמשכים על אזורים חמצן נמוכה של מים חופי האוקיינוס ​​הפתוח. דאו נתמכה על ידי מלגות של NSERC, Killam ויסוד טולה מרכז מימנה עבור גיוון התפתחות חיידקים.

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הגדרת מנגנון סינון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לפני סינון, תחילה למחות כל התעבות של בקבוקים מלאים ולשקול אותם </li><li style=";text-align:right;direction:rtl"> הצב בן החיטוי בצד אחד של המשאבה, ואת בקבוק פסולת מאידך. </li><li style=";text-align:right;direction:rtl"> אז במקום בקבוק מלא מדגם לפח החיטוי, אך לא להסיר את מכסה הבקבוק עדיין. </li><li style=";text-align:right;direction:rtl"> במעבר דרך צינור המשאבה peristaltic כך בסוף luer של צינורות נמצא הבקבוק. </li><li style=";text-align:right;direction:rtl"> בעזרת פינצטה, להסיר את הכותנה מן פיפטה סטרילית 25 מ&quot;ל, ולצרף את טפטפת לקצה השני של הצינור. </li></ol></li><li style=";text-align:right;direction:rtl"> סינון של מי ים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי להתחיל סינון מי ים, לפתוח את הבקבוק ולהכניס את פיפטה. הפעל את המשאבה ולקבוע את מהירות הזרימה 1. צפה על מנת להבטיח כי המים נשאב בכיוון הנכון. </li><li style=";text-align:right;direction:rtl"> אפשר ~ 100 מ&quot;ל מים לעבור דרך צינורות כולו. </li><li style=";text-align:right;direction:rtl"> כבה את המשאבה ואת לצרף לסנן Sterivex ל מתאים luer זכר בסוף חופשית של הצינור. </li><li style=";text-align:right;direction:rtl"> המשך עד אין סינון מים נשאר הבקבוקים. </li></ol></li><li style=";text-align:right;direction:rtl"> אחסון של מסננים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> כדי לאחסן את המסננים, תחילה לגרש כל המים שנותרו עם מזרק 3-60 מ&quot;ל. </li><li style=";text-align:right;direction:rtl"> חותם את החלק התחתון של המסנן Sterivex עם Parafilm. בעזרת פיפטה, מוסיפים למאגר תמוגה 1.8ml לסינון, שמירה ~ 200 ul שטח במסנן עבור תוספת מאוחרת של חומרים כימיים. </li><li style=";text-align:right;direction:rtl"> לאחר מכן, חותם את החלק העליון של המסנן Sterivex עם חתיכה קטנה של Parafilm, ואת התווית מסננים עם התאריך וזיהוי המדגם. </li><li style=";text-align:right;direction:rtl"> חנות מסננים מראש שכותרתו צינורות 50 מ&quot;ל בז ב -80 ° C </li></ol></li><li style=";text-align:right;direction:rtl"> ניקוי <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנס את פיפטה 25 מ&quot;ל לתוך הבקבוק המכיל 0.1% HCl, ולעבור על המשאבה. </li><li style=";text-align:right;direction:rtl"> לאחר לשטוף את HCl, לשטוף בצינור מים autoclaved. לבסוף, למדוד ולתעד המוני של בקבוקי 1 ליטר ריק עכשיו, כדי לחשב את נפח המים עברו דרך המסנן. </li></ol></li></ol> נציג תוצאות: לכל 1 ליטר מים מדגם התשואות one Sterivix מסנן, אשר מכיל ביומסה גדולה 0.22 מיקרומטר. זה מאוחסן חיץ תמוגה להפקת DNA ניתוח עתידיים. התוצאה הסופית כוללת מסנן Sterivix אחד לכל בקבוק 1 ליטר מאוחסנים חיץ תמוגה ומוכן להפקת DNA. המסנן מכיל ביומסה גדולה 0.22 מיקרומטר. מסננים ניתן לאחסן -80 ° C לפני מיצוי עדנה.

The authors have nothing to disclose.

זה הכרחי כדי לנגב את ספסלי משאבות במגבת לחה לאחר השימוש כדי להסיר כל עקבות המתמשכת של מי מלח שאחרת לשיתוק ציוד. למרות ההליך הוא פשוט, הדרישה זמן יכול להיות נרחב תלוי בריכוז של ביומסה או פסולת במדגם להיות מסונן.

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Peristaltic pump</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>77913-20, 77200-62, 77200-04</td>
<td>&nbsp;</td>
<tr>
<td>Tygon LFL tubing</td>
<td>&nbsp;</td>
<td>Masterflex (Cole Palmer)</td>
<td>06429-24</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Hose clamps</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>0683204</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>1/4&#x201D; x 3/16&#x201D; pipe adaptors</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31702-20</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Polypropylene prefilter housing</td>
<td>&nbsp;</td>
<td>ADVANTEC</td>
<td>501200</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>Metal luer fitting (Hose end to Male luer lock)</td>
<td>&nbsp;</td>
<td>Cole Palmer</td>
<td>31507-27</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>2&#x201C; Magnets</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>3 port carboy caps</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For filtration apparatus</td>
</tr>
<tr>
<td>GV 0.22 &mu;m STERIVIX filters</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>SVGV01R5</td>
<td>&nbsp;</td>
<tr>
<td>47 mm diameter 2.7 &mu;m GF/D filters</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>1823047</td>
<td>&nbsp;</td>
<tr>
<td>Lysis buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>0.75 M sucrose, 40 mM EDTA, 50 mM Tris (pH 8.3)</td>
</tr>
<tr>
<td>0.1 % HCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>
<tr>
<td>Autoclaved water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>For post-filtration rinse</td>
</tr>		</tbody>
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small volume (1-3l) filtration of coastal seawater samples

עבודה מתחיל עם איסוף המים ימית החוף למען הקהילה חיידקים במורד הזרם, מנתח גז מזין עקבות. לקבלת דוגמיות הפגנה של היום נאספו סיפון ההפעלה ג&#39;ון סטריקלנד HMS ב Inlet Saanich. זה מסמכים וידאו נפח קטן (~ 1 L) סינון של ביומסה של חיידקים מעמודת המים. הפרוטוקול הוא הרחבה של פרוטוקול גדול הדגימה נפח שתוארו קודם לכן, עם הבדל אחד משמעותי: כאן, אין שום צעד סינון מראש, ולכן כל המעמדות גודל של ביומסה נאספים עד 0.22 מיקרומטר לסנן חתוכים. דוגמאות שנאספו בדרך זו הם אידיאליים עבור ניתוח חומצות גרעין. הצעדים הגדרת, סינון, ו לנקות כל אחד לקחת על 20-30 דקות. אם באמצעות שתי משאבות peristaltic בו זמנית, עד 8 דגימות ניתן לסנן באותו זמן. כדי למנוע היווצרות biofilm בין טיולים הדגימה, כל הציוד סינון חייב להיות שטוף עם HCl לדלל במים deionized ו autoclaved מיד לאחר השימוש.

Watch this Scientific Journal Video about Small Volume 1-3L Filtration of Coastal Seawater Samples at JoVE.com

Small Volume 1-3L Filtration of Coastal Seawater Samples

זה מסמכים וידאו נפח קטן (~ 1 L) סינון של ביומסה של חיידקים מעמודת המים.

נפח קטן (1-3L) סינון של דגימות מי ים החוף

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For today s ...

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Small Volume (1-3L) Filtration of Coastal Seawater Samples

Introduction to Separation Methods

SCIENTISTS IN ACTION

11.1K Views.  University of British Columbia - UBC. זה מסמכים וידאו נפח קטן (~ 1 L) סינון של ביומסה של חיידקים מעמודת המים.

Video: Small Volume 1-3L Filtration of Coastal Seawater Samples

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

מי ים דגימה אוסף

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Large Volume (20L+) Filtration of Coastal Seawater Samples

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column. Two 20L samples can be filtered simultaneously using a single pump unit equipped with four rotating heads. Filtration is done in the field on extended trips, or immediately upon return for day trips. It is important to record the amount of water passing through each sterivex filter unit. To prevent biofilm formation between sampling trips, all filtration equipment must be rinsed with dilute HCl and deionized water and autoclaved immediately after use. This procedure will take approximately 5 hours plus an additional hour for clean up.

Large Volume 20L+ Filtration of Coastal Seawater Samples

This video documents large volume (&ge;20 L) filtration of microbial biomass, ranging between 0.22&mu;m and 2.7&mu;m in diameter, from the water column.

נפח גדול (20L +) סינון של דגימות מי ים החוף

The workflow begins with the collection of coastal marine waters for downstream microbial community, nutrient and trace gas analyses. For this method, ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

Quantitation של γH2AX מוקדים של דגימות רקמה

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

מדידה אבסולוטית קוונטית תשואה של דוגמאות אבקת

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

דור מצע לEndonucleases של מערכות CRISPR / קאש

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

שיטת תפוקה גבוהה למדידה של קצב סינון הגלומרולרי בעכברים בהכרה

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Rapid Isolation And Purification Of Mitochondria For Transplantation By Tissue Dissociation And Differential Filtration

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator&#8217;s standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 1010 viable and respiration competent mitochondria from 0.18 &#177; 0.04 g (wet weight) tissue sample.

A method for rapid isolation of mitochondria from mammalian tissue biopsies is described. Rat liver or skeletal muscle preparations were homogenized with a commercial tissue dissociator and mitochondria were isolated by differential filtration through nylon mesh filters. Mitochondrial isolation time is &#60;30 min compared to 60 - 100 min using alternative methods.

בידוד מהיר וטיהור של מיטוכונדריה להשתלה על ידי דיסוציאציה רקמות ודיפרנציאל סינון

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to ...

Physiology Lab Demonstration: Glomerular Filtration Rate in a Rat

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function in health and disease. GFR is measured as the steady state renal clearance of inulin which is filtered at the glomerulus, but not secreted or reabsorbed along the nephron. The fractional excretion of Na and K can be determined from the concentration of Na and K in plasma and urine. The renal clearance of inulin can be demonstrated in an anesthetized animal which has catheters in the femoral artery, femoral vein and bladder. The equipment and supplies used for this procedure are those commonly available in a research core facility, and thus makes this procedure a practical means for measuring renal function. The purpose of this video is to demonstrate the procedures required to perform a lab demonstration in which renal function is assessed before and after a diuretic drug. The presented technique can be utilized to assess renal function in rat models of renal disease.

The purpose of this protocol is to demonstrate the principles and techniques for measuring and calculating glomerular filtration rate, urine flow rate, and excretion of sodium and potassium in a rat. This demonstration can be used to provide students with an overall conceptual understanding of how to measure renal function.

הפגנת מעבדה לפיזיולוגיה: גלומרולרי סינון שיעור בחולדה

Measurements of glomerular filtration rate (GFR), and the fractional excretion of sodium (Na) and potassium (K) are critical in assessing renal function ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

מדידת לולאות נפח לחץ העכבר

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

בידוד סינון של חומצות גרעין: שיטת הפקת DNA פשוטה ומהירה

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...