DNA Dilution Calculator
Enter DNA concentrations (ug/ul) for up to 10 samples and specify the final volume (ul) to calculate dilution volumes.
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Showing posts with label molecular biology. Show all posts
Showing posts with label molecular biology. Show all posts
Mouse Age Calculation - Excel and web calculator
This document explains two methods for calculating the age of a mouse. The first method involves using Excel to perform the calculations, while the second method utilizes an online web tool calculator for a more straightforward approach.
Mouse Age Calculation
Mouse Age Calculation in Excel
Follow these steps to calculate the age of a mouse in Excel:
Create a New Excel Document:
- Open Excel and create a new document.
Set Column Headers:
- In cell A1, type "Birthdate."
- In cell B1, type "Current Date."
- In cell C1, type "Age (Days)."
- In cell D1, type "Age (Weeks)."
Enter Dates:
- In cell A2, input the birthdate of the mouse (e.g., "2023-01-15").
- In cell B2, use the following formula to automatically input the current date:
=TODAY()
Calculate Age:
- In cell C2, enter the following formula:
=B2-A2 - In cell D2, use the following formula to convert age (days) into weeks (weeks old):
=INT(C2/7)
Check the Results:
- Cells C2 and D2 will display the current age of the mouse in days and weeks, respectively.
You can now calculate the age of a mouse in Excel using these steps. If you have any further questions or need assistance, feel free to ask.
Mouse Age Calculation in web calculator
How to Use
1. Enter the birthdate of the mouse in the input field above.
2. Click the "Calculate" button to determine the mouse's age.
3. The age of the mouse in days and weeks will be displayed below.
Enter the birthdate of the mouse:
Star Activity: Understanding and Solutions
During the process of performing restriction enzyme digestion for Sanger sequencing, an unusual phenomenon called 'Star Activity' was observed. Typically, when DNA is processed using two restriction enzymes, each of which is known to exist in DNA, two bands should appear—one for the fragment of DNA cut by each enzyme and the other for the remaining DNA. However, in the case of DNA treated with restriction enzymes, an unexpected phenomenon was observed. Instead of the expected two bands, three to four DNA fragments are being identified.
Understanding and Addressing Star Activity in Restriction Enzyme Digestion
What is Star Activity?
"Star Activity" refers to the phenomenon where a restriction enzyme cuts DNA at sites other than its specific recognition sequence. This non-specific cleavage can distort experimental results and make accurate analysis challenging.
Causes of Star Activity:
Star Activity can be attributed to a variety of factors, including suboptimal buffer conditions, non-specific binding, and the presence of certain ions and solvents:
Suboptimal Buffer Conditions:
Star Activity can occur when buffer conditions, such as temperature variations, improper pH levels, and inadequate ion concentrations, are not optimized for the restriction enzyme used. These suboptimal conditions can lead to deviations from expected results and challenges in accurate DNA analysis.
Non-Specific Binding:
The restriction enzyme may bind to regions of DNA outside its specific recognition sequence, leading to cleavage at unconventional locations. This non-specific binding can be influenced by factors like DNA quality and enzyme concentration.
Presence of Certain Ions and Solvents:
Star Activity may also result from the presence of divalent cations other than Mg2+, organic solvents like ethanol, and high glycerol concentrations (exceeding 5% v/v) in the reaction mixture. These conditions can disrupt the enzyme's specificity and contribute to non-specific cleavage.
Addressing Star Activity requires a thorough understanding of these causes and appropriate adjustments to the experimental conditions and buffer systems to minimize its impact.
Solutions to Prevent Star Activity
To prevent Star Activity and optimize the use of restriction enzymes, consider the following approaches:
1. Research the Enzyme
Conduct literature research on the specific restriction enzyme you plan to use to understand its recognition site and conditions for optimal activity.
2. Optimize Buffer Conditions
Carefully fine-tune buffer conditions, including temperature, ion concentration, and pH, to create an optimal enzymatic environment, reducing the likelihood of non-specific cleavage in the DNA.
For instance, if you are working with the restriction enzyme EcoRI, you might optimize the buffer conditions by testing different pH levels (e.g., pH 7.4, 7.6, and 7.8) to find the pH at which EcoRI shows the least Star Activity while still efficiently cutting the target DNA. This fine-tuning can help ensure more accurate and reliable DNA cleavage.
3. Minimize Non-Specific Binding
Minimizing Non-Specific Binding involves reducing the likelihood of the restriction enzyme binding to unintended DNA sequences.
For instance, when working with the restriction enzyme HindIII, which recognizes the specific sequence 5'-AAGCTT-3', you can minimize non-specific binding by using purified DNA samples free from sequences resembling 'AAGCTT' and adjusting the enzyme concentration to ensure that it predominantly binds to the intended recognition site. This ensures that the enzyme cuts the target DNA accurately, reducing the chance of non-specific cleavage and Star Activity.
4. Select the Right Enzyme
Empirically monitor or prevent Star Activity by choosing the most suitable restriction enzyme from a variety of options.
5. Avoid Rapid Temperature Changes
After PCR or restriction enzyme treatment, avoid rapid temperature changes, thoroughly cool the sample, and then proceed with analysis.
Minimizing Star Activity requires adjusting experimental conditions and selecting the appropriate restriction enzyme. Monitoring and addressing any Star Activity that occurs during experiments is crucial for successful DNA analysis.
In summary, Star Activity in restriction enzyme digestion is a phenomenon that can lead to unexpected DNA cleavage, causing deviations from anticipated results in molecular biology experiments. It can be triggered by factors such as suboptimal buffer conditions, non-specific binding, and the presence of certain ions and solvents. To mitigate Star Activity and ensure accurate DNA analysis, researchers should carefully optimize buffer conditions, control non-specific binding, and be mindful of the specific reaction conditions. Addressing these factors is crucial for reliable and reproducible results in molecular biology experiments involving restriction enzymes.
Competent Cells Transformation: A Step-by-Step Guide
In this post, we present a concise overview of cell transformation protocols. Competent Cells transformation is a pivotal process in molecular biology and genetic engineering, and we'll provide essential information for successful experiments.
Competent Cells Transformation
: A Step-by-Step Guide
Here, we have summarized the protocols for One Shot® TOP10 Competent Cells and NEB® 5-alpha Competent E. coli (High Efficiency). Each protocol refers to the procedures included in the corresponding product.
One Shot® TOP10 Competent Cells
1. Centrifuge the vial(s) containing the ligation reaction(s) briefly and place on ice.
2. Thaw, on ice, one 50 μL vial of One Shot® cells for each ligation/transformation.
3. Pipet 1–5 μL of each ligation reaction directly into the vial of competent cells and mix by tapping gently. Do not mix by pipetting up and down. The remaining ligation mixture(s) can be stored at −20°C.
4. Incubate the vial(s) on ice for 30 minutes.
5. Incubate for exactly 30 seconds in the 42°C water bath. Do not mix or shake.
6. Remove vial(s) from the 42°C bath and place them on ice.
7. Add 250 μL of pre-warmed S.O.C medium to each vial. S.O.C is a rich medium; sterile technique must be practiced to avoid contamination.
8. Place the vial(s) in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s). Shake the vial(s) at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.
9. Spread 20–200 μL from each transformation vial on separate, labeled LB agar plates. The remaining transformation mix may be stored at 4°C and plated out the next day, if desired.
10. Invert the plate(s) and incubate at 37°C overnight.
11. Select colonies and analyze by plasmid isolation, PCR, or sequencing.
NEB® 5-alpha Competent E. coli (High Efficiency)
1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.
Simple Timer
Timer Web App
How to Use
You can use this web app to set and run a simple timer. Follow these steps:
- Enter your desired hours, minutes, and seconds in the input fields.
- Click the "Set Timer" button to configure the timer.
- The timer will start counting down until the specified time elapses.
- When the timer expires, a browser notification will be displayed.
Note: To receive notifications in your browser, you need to grant notification permissions. When the notification permission request pops up, select "Allow."
00:00:00
Nuclear protein isolation protocol (with kit AB219177 Abcam)
The ab219177 Nuclear Extract Kit is a powerful tool for extracting nuclear proteins from mammalian cells or tissues in just 45 minutes. These nuclear proteins are essential for various applications, including western blotting and nuclear enzyme assays. In this blog post, we'll guide you through the protocol to make the process easy to understand and follow.
Nuclear protein isolation protocol
Using Nuclear Extraction Kit (AB219177, Abcam)
Materials You'll Need:
Before you start, gather the following materials:
The ab219177 Nuclear Extract Kit
1X Phosphate-buffered saline (PBS)
Trypsin/EDTA solution
Double-distilled water (ddH2O)
1.5 mL and 15 mL plastic tubes
Benchtop microcentrifuge
Centrifuge for 15 mL tubes
Sonicator
Step-by-Step Protocol:
Step 1: Preparation
◈ Ensure that PBS is at 4°C and store it on ice.
◈ Cool the benchtop microcentrifuge to 4°C.
◈ If you plan to use the extracts for enzyme activity assays, do not add Protease Inhibitor Cocktail to any buffers or fractions.
Step 2: Buffer Preparation
◈ For each extraction, transfer 500 µL each of Cytoplasmic Extraction Buffer, Nuclear Extraction Buffer, and Nuclear Lysis Buffer into clean 1.5 mL microcentrifuge tubes and keep them on ice.
◈ To each tube, add 2.5 µL of 200X Protease Inhibitor Cocktail and 2.5 µL of 200X DTT. Keep the tubes on ice until needed.
Cytoplasmic Extraction Buffer (++)
Nuclear Extraction Buffer (++)
Nuclear Lysis Buffer (++)
Step 3: Cell Preparation
◈ For adherent cells, grow cells to 70-80% confluence and remove the growth medium.
◈ Wash the cells with room temperature PBS twice.
◈ For suspension cells, grow cells to 2 x 106/mL.
◈ For tissues, weigh the tissue and cut it into small pieces for homogenization.
◈ Wash tissues twice with ice-cold PBS.
Step 4: Cell/Tissue Processing
◈ Follow specific instructions based on cell type (adherent, suspension, or tissues) for the next steps. These include resuspending the cells, centrifugation, and preparation for extraction. Please refer to the procedure for adherent cells inside the blue box below.
◈ Centrifuge for 5 minutes at 1,000 rpm (4°C) and discard the supernatant.
◈ Wash cells with 10 mL of ice-cold PBS by centrifugation for 5 minutes at 1,000 rpm (4°C) and discard the supernatant.
Step-by-Step Protocol for Adherent Cells:
a. Grow Adherent Cells
Cultivate your adherent cells on a culture plate or flask until they reach 70-80% confluency.
Remove the growth medium from the plate.
b. Wash the Cells
Wash the cells twice with room temperature PBS (Phosphate-buffered saline).
Carefully discard the PBS after each wash.
c. Collect the Cells
For every 20 cm2 of cell growth area, add 1 mL of room temperature PBS. (3 mL for 100 mm plate)
Use a cell scraper to gently detach the cells from the surface of the culture plate. Ensure all cells are in suspension.
d. Optional: Use Trypsin/EDTA
Alternatively, you can use trypsin/EDTA solution for detachment.
Dispense enough trypsin/EDTA solution to completely cover the monolayer of cells.
Incubate the cells in a 37°C incubator for approximately 2 minutes or until they detach from the surface.
Once detached, the cells will appear rounded.
e. Protect the Cells
Immediately after trypsinization, add serum or media containing serum to the cell suspension.
This helps protect the cells from any potential damage caused by the trypsin activity.
Note: It's important to be aware that the process of trypsinization may have an impact on the cellular pathway you are studying, so consider this when planning your experiments.
Step 5: Extraction of Cytoplasmic Proteins
◈ Resuspend the cell pellet in Cytoplasm Extraction Buffer (+/+) and transfer to a 1.5 mL tube.
◈ Vortex briefly and incubate cells on ice for 10 minutes.
◈ Vortex briefly again and centrifuge for 3 min at 1,000 g (4°C).
◈ Trasfer the supernatant (cytoplasmic protein extract) to new ice-cold 1.5 mL tube and keep both the pellet and the supernatant on ice.
Step 6: Extraction of Soluble Nuclear Proteins
◈ Resuspend the pellet from the previous step in Nuclear Extraction Buffer (++).
◈ Vortex briefly and incubate cells on ice for 15 minutes (with vortex every 5 min).
◈ Vortex briefly again and centrifuge for 3 min at 5,000 g (4°C).
◈ Trasfer the supernatant (soluble nuclear proteins "Nuclear Extract 1") to new ice-cold 1.5 mL tube and keep both the pellet and the supernatant on ice.
Step 7: Extraction of Insoluble Nuclear Proteins
◈ Resuspend the pellet from step 6 in Nuclear Lysis Buffer (++).
◈ Sonicate (low) the sample on ice to obtain "Nuclear Extract 2", which contains remaining insoluble nuclear proteins.
Step 8: Protein Quantification and Analysis
◈ Measure the protein concentration of the extracted fractions (BCA assay).
◈ Use the fractions immediately or aliquot and freeze at -80°C for future use.
Conclusion:
Using the ab219177 Nuclear Extract Kit, you can efficiently extract nuclear proteins from mammalian cells and tissues. This protocol simplifies the process into clear steps, making it accessible for your research needs.
[EasyTools] PCR Mixture Calculator
PCR Mixture Calculator
Calculation Result:
Typically, the cDNA concentration used in qPCR falls within the range of 10 ng/μL to 100 ng/μL. This range is commonly used in various experiments, but it may be adjusted based on the experiment's objectives and sample types.
This calculator is based on a cDNA volume of 1ul. PCR mixture 19 ul + cDNA 1 ul
Manual RNA Isolation Protocol
In the world of molecular biology, RNA isolation is a fundamental step in studying gene expression and unraveling the mysteries of life at the molecular level. If you're new to this field, fear not! This blog post serves as your stepping stone into the fascinating realm of manual RNA isolation. We'll walk you through each step of the process, providing clear and concise instructions to ensure your success. Whether you're a curious novice or a seasoned scientist looking for a refresher, this guide will equip you with the knowledge and skills to extract high-quality RNA for your research needs. Let's dive in!"
Materials Needed:
- Tissue or cells
- TRIzol™ Reagent
- Chloroform
- Isopropanol
- 75% ethanol
- RNase-free water
- Microcentrifuge tubes
- Centrifuge
- Pipettes and tips
- RNase-free gloves
Procedure:
1.Sample Preparation
- Begin with your tissue or cells. If you have tissue, homogenize it in a suitable buffer. If you have cells, proceed to the next step.
2.Cell Lysis
- Add TRIzol reagent (1 mL per 1 x 10^6 cells or 50-100 mg tissue) to the sample.
- Mix thoroughly and incubate for 5 minutes at room temperature.
3.Phase Separation
- Add chloroform (0.2 mL per 1 mL TRIzol used).
- Shake vigorously for 15 seconds.
- Incubate for 2-3 minutes at room temperature.
- Centrifuge at 12,000 x g for 15 minutes at 4°C.
4.RNA Precipitation
- Carefully transfer the aqueous phase (upper layer) to a new tube.
- Add an equal volume of isopropanol to the aqueous phase.
- Mix and incubate at room temperature for 10 minutes.
- Centrifuge at 12,000 x g for 10 minutes at 4°C.
5.Washing and Pelleting RNA
- Carefully remove the supernatant.
- Wash the RNA pellet with 75% ethanol.
- Centrifuge at 7,500 x g for 5 minutes at 4°C.
- Carefully remove the ethanol and air-dry the RNA pellet for 5-10 minutes.
6.RNA Resuspension
- Resuspend the RNA pellet in RNase-free water.
7.Assess RNA Quality and Quantity
- Measure the RNA concentration using a spectrophotometer.
- Verify RNA quality through gel electrophoresis or a Bioanalyzer.
Precautions When Handling RNA
it's essential to handle RNA carefully to avoid degradation and contamination. Here are some key precautions to keep in mind when working with RNA:
1. Maintain a Sterile Environment:
Work in a clean and dedicated RNA-free workspace.
Use RNase-free reagents, equipment, and labware.
Wear clean lab coats, gloves, and change them regularly.
2. Prevent RNase Contamination:
RNases are enzymes that can quickly degrade RNA. Avoid touching surfaces with bare hands.
Use RNase inhibitors in your buffers.
Autoclave or use commercial RNase decontamination reagents for labware.
3. Minimize RNA Exposure to Oxygen:
RNA is sensitive to oxidation. Keep samples on ice or at -80°C when not in use.
Use RNase-free, sterile, and aerosol-resistant pipette tips.
4. Quick Sample Handling:
Keep sample handling times as short as possible.
Avoid unnecessary freeze-thaw cycles.
5. Precipitate RNA in Cold Isopropanol:
Ensure that isopropanol used for RNA precipitation is stored at -20°C or colder.
Perform RNA precipitation steps at -20°C or colder.
6. Gentle Mixing:
When resuspending RNA pellets, vortex gently or pipette up and down gently to avoid shearing.
7. Monitor RNA Quality:
Check RNA quality and integrity using gel electrophoresis, Bioanalyzer, or similar methods.
8. Store RNA Properly:
Store RNA samples at -80°C for long-term storage.
Use RNase-free tubes and ensure proper sealing to prevent sample contamination.
9. Use RNA Gloves:
Use gloves specifically designed for RNA work to minimize skin contact.
10. Plan for Contingencies:
Have backup samples in case of unexpected RNA degradation.
In conclusion, working with RNA demands meticulous care and attention to detail. By following the precautions outlined in this guide, you can ensure the integrity of your RNA samples, setting the stage for successful experiments and accurate results. Remember, RNA is a delicate molecule, but with the right precautions, you can harness its power to unlock the secrets of genetic information. Happy RNA handling, and may your research endeavors be fruitful!
Mastering RNA Handling: Essential Lab Guidelines
Working with RNA in the laboratory demands precision and care. Whether you're new to the world of molecular biology or looking to refresh your knowledge, this quick guide will provide essential tips for RNA handling and precautions to ensure successful experiments. Let's dive in!
Mastering RNA Handling
Handling and Precautions for RNA
◼ Reagents used for RNA preparation and analysis should be kept separate from other reagents.
◼ During work, avoid unnecessary talking, wear a mask, and use clean disposable plastic gloves. Use dedicated RNA workstations and benches to minimize contamination.
◼ Prepare reagent solutions for RNA work with 0.1% DEPC-treated water and autoclave them before use. If autoclaving is not possible due to sensitive components in the reagents, prepare solutions using sterilized equipment and water, followed by filtration sterilization.
Method for preparing 0.1% DEPC-treated water:
Add Diethyl pyrocarbonate (DEPC) to distilled water at a concentration of 0.1% (v/v). Stir the solution at room temperature for one night (or 12 hours at 37°C). Then, autoclave the solution at 120°C for 30 minutes to remove DEPC completely.
Caution: DEPC is a chemical reagent used as an RNase inhibitor, but it is carcinogenic, so handle it with care.
◼ Most disposable sterile plasticware available in the market is RNase-free and can be used directly for experiments. However, autoclave standard microcentrifuge tubes and micro pipet tips before use. If using glassware or spatulas, autoclave them at 180°C for at least 1 hour. If autoclaving is not possible, soak them in 0.1% DEPC solution at room temperature for one night (or 12 hours at 37°C), then autoclave before use.
Preparation of RNA Samples
◼ In many experiments, high-purity RNA samples are required. Especially for DNA chip analysis, impurities such as carbohydrates or proteins can interfere with the reaction or lead to high background signals. Preventing contamination with genomic DNA is also crucial.
◼ For tissues and cells, RNA should be extracted immediately after sample collection. If immediate extraction is not possible, store samples at -80°C or in liquid nitrogen.
Preparation of Total RNA
Use methods such as cesium chloride density gradient centrifugation or Guanidine thiocynate phenol chloroform (AGPC) extraction, or commercially available RNA purification reagents.
Purification of Poly(A)+ RNA
Poly(A)+ RNA is commonly isolated from total RNA using methods involving Oligo(dT) Cellulose or similar techniques.
Purity and Concentration Assessment of RNA
◼ In most cases, the purity of RNA greatly affects experimental results. Therefore, it is essential to assess RNA purity before use.
◼ Purity Assessment of Total RNA by Gel Electrophoresis
Denature 1-2 µg of total RNA (65°C for 10 minutes) and electrophorese it on a 1% agarose gel (TBE buffer). Intact total RNA should show two distinct ribosomal RNA bands (28S and 18S in eukaryotes, 23S and 16S in prokaryotes) in a ratio of approximately 2:1. If ribosomal RNA bands are smeared, it may indicate RNA degradation due to RNase contamination. If there are larger bands than 28S (or 23S), it suggests the presence of genomic DNA contamination, and DNase I (RNase-free) treatment should be performed to remove genomic DNA.
◼ Purity and Quantification of Total RNA and Poly(A)+ RNA by UV Absorbance
Measure the absorbance at 260nm and 280nm to assess purity and concentration of total RNA (or Poly(A)+ RNA).
A260/A280 ratio:
A ratio of 1.8-2.1 indicates low protein contamination and high-purity RNA samples.
A ratio below 1.7 is not suitable for DNA chip experiments.
Calculate RNA concentration using A260=1, which corresponds to 40 µg/ml.
Example: If you have 100 µl of RNA sample, and the absorbance at A260 is 0.65, then
RNA concentration = 40 µg/ml x A260 x dilution factor
= 40 x 0.65 x 50
= 1300 µg/ml
Total RNA amount = concentration x sample volume (ml)
= 1300 x 0.1
= 130 µg
Precise analysis of RNA, including total RNA and mRNA, can be achieved through nucleic acid gel electrophoresis and automated detection devices for accurate results.
Concentration Calculations Made Easy
New to chemistry and lab work? Understanding concentration measurements and solution units is essential for success. In this beginner's guide, we'll simplify these concepts, making them accessible and practical for your experiments. Join us as we explore the basics and equip you with the knowledge to excel in your scientific journey.
Concentration Calculations Made Easy
Molar Concentration (M)
: Molar concentration represents the number of moles (M) of solute present in 1 liter of solution. It is calculated as the ratio of the number of moles to the volume of the solution in liters.
"grams (g) = molecular weight × M (molar concentration) × L (volume)"
Example 1: If you want to prepare a 1 M NaCl solution with a volume of 1 liter, you can calculate the amount of NaCl needed as follows:
First, consider the molecular weight of NaCl, which is 58.44 g/mol.
Take into account the desired molar concentration, which is 1 M (1 mol/L).
The required amount of NaCl is calculated as follows:
Required NaCl (g) = Molecular Weight (g/mol) × Molar Concentration (mol/L) × Volume (L)
Required NaCl (g) = 58.44 g/mol × 1 mol/L × 1 L
Performing the calculation yields a required amount of 58.44 g of NaCl.
So, to prepare a 1 M NaCl solution with a volume of 1 liter, you would need 58.44 grams of NaCl.
EasyTools - Solution Dilution Calculator
Normality (N)
: Normality is the equivalent weight of a solute (in grams) per liter of solution. An equivalent is the amount of a substance that can either gain or lose one mole of electrons in a chemical reaction. It is used primarily in acid-base reactions.
"grams (g) = molecular weight / valence × N (normality) × L (volume)"
Example 1: If you want to prepare 1 L of a 1 N AgNO3 solution, the required amount of AgNO3 in grams can be calculated as follows:
Molecular weight of AgNO3 = 170
Valence = 1
Using the formula: grams (g) = molecular weight / valence × N (normality) × L (volume)
X g = 170/1 x 1 x 1 = 170 g
Therefore, by dissolving 170 grams of AgNO3 in 1 L of water, you'll obtain a 1 N AgNO3 solution.
When the valence is "1," the Molarity (M) and Normality (N) concentrations are the same.
Percentage Concentration
: Percentage concentration expresses the amount of solute as a percentage of the total solution weight or volume.
- Weight/Weight (% w/w): The weight of solute in grams per 100 grams of solution.
- Volume/Volume (% v/v): The volume of solute in milliliters per 100 milliliters of solution.
- Weight/Volume (% w/v): The weight of solute in grams per 100 milliliters of solution.
- Volume/Weight (% v/w): The volume of solute in milliliters per 100 grams of solution.
Parts Per Million (ppm)
: Parts per million is a unit for expressing very low concentrations. It represents the number of milligrams (mg) of solute per liter of solution.
Note: ppm stands for "parts per million," indicating one part in a million, or 1/1,000,000.
| Concentration Unit | Calculation Method | |
|---|---|---|
| Molar Concentration (M) | Moles of solute / Volume (liters) = mol/L | |
| Normality (N) | Equivalent weight of solute (g) / Volume (liters) = N | N = M * Equivalent Factor* |
| Percentage (% w/w) | Mass of solute (g) / Total mass of solution (g) x 100 = % w/w | |
| Percentage (% v/v) | Volume of solute (mL) / Total volume of solution (mL) x 100 = % v/v | |
| Percentage (% w/v) | Mass of solute (g) / Total volume of solution (mL) x 100 = % w/v | |
| Percentage (% v/w) | Volume of solute (mL) / Total mass of solution (g) x 100 = % v/w | |
| Parts Per Million (ppm) | Mass of solute (mg) / Total volume of solution (liters) = ppm | ppm = 1000 * M (mg/L) |
*Equivalent Factor (Normality - N)
: In chemistry, the Equivalent Factor, also known as Equivalent Weight, plays a vital role in normality (N) calculations. It represents the weight of a substance that can either gain or lose one mole of electrons or react with one mole of hydrogen ions (H⁺) in a chemical reaction.
Here's a simplified explanation of Equivalent Factors:
- For monoprotic acids and bases (e.g., HCl and NaOH), the Equivalent Factor is equal to the molar mass of the substance.
- For diprotic acids or bases (e.g., H2SO4), the Equivalent Factor is half the molar mass because one mole of the substance can neutralize two moles of H⁺ or OH⁻ ions.
- For polyprotic acids or bases, the Equivalent Factor is adjusted accordingly based on the reaction stoichiometry.
This concept is particularly valuable in normality (N) calculations, where it ensures that the concentration of substances in a solution is measured in equivalents per liter, accounting for their specific reactivity in chemical reactions. This knowledge is essential for accurate titrations and understanding the behavior of substances in various chemical processes.
Mouse Genotyping
Mouse Genotyping protocol
Step 1: Gather Materials and Reagents
- Make sure you have all the necessary materials and reagents ready for the genotyping process. This includes the tissue samples (tail snips, ear punches, or other tissues), Extraction Reagent, Stabilization Buffer, PCR reagents, and agarose gel.
- AccuStart™ II Mouse Genotyping Kit (Cat. No. 95135-100; Size: 100 reactions; Quantabio)
Step 2: Prepare Tissue Samples
- Depending on the type of tissue samples you have (tail snips, ear punches, or other tissues), determine the appropriate volume of Extraction Reagent needed. Refer to the table provided earlier for guidance on sample sizes and Extraction Reagent volumes.
- Ensure that tissue samples are small and completely submerged in Extraction Reagent.
| Sample Type | Sample Size | Extraction Reagent Volume | Comments |
|---|---|---|---|
| Tail Snips | 2 mm | 100 μL | Fresh or frozen tail snips can be used. Use proportionally more Extraction Reagent with tail snips larger than 2 mm. Samples will appear to remain intact and will not dissolve in Extraction Reagent after heating. |
| Ear Punches | 2 mm | 50 μL | Ensure that the ear punches are completely submerged in Extraction Reagent. |
| Other Tissues | 5 mg | 100 μL | Tissue samples should be small and completely submerged in Extraction Reagent. |
Step 3: DNA Extraction
- Add the tissue samples to the appropriate volume of Extraction Reagent as calculated in Step 2.
- Heat the samples to 95°C for 30 minutes. Note that the tissue samples will not dissolve and will appear intact after incubation.
- Cool the samples to room temperature.
- Add an equal volume of Stabilization Buffer to the cooled samples. This serves as a safe stopping point.
- Extracts can be stored at 4°C for several weeks or at -20°C for several months.
Step 4: PCR Reaction Setup
- Use up to 2.5 μL of the DNA extract in a 25 μL PCR reaction. Depending on the sample size and extraction conditions, you may need to dilute the extract in water or TE buffer for optimal PCR results.
- Follow your standard PCR reaction setup protocols for amplifying the DNA regions of interest.
Step 5: Gel Electrophoresis
- Load 5 or 10 μL (or as appropriate) of the PCR products on an agarose gel.
- Perform gel electrophoresis to visualize and confirm the presence of the desired DNA fragments.
Additional Notes:
The protocol can be optimized for specific applications by adjusting incubation times or omitting the addition of Stabilization Buffer if needed.
It's recommended to use PCR tubes or multi-well plates and incubate in a thermal cycler with a heated lid to prevent sample condensation.
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