🧪 Resuspension Calculator
Calculate the volume needed to resuspend your dry oligos to a desired concentration.
Calculate the volume needed to resuspend your dry oligos to a desired concentration.
Enter DNA concentrations (ug/ul) for up to 10 samples and specify the final volume (ul) to calculate dilution volumes.
| Sample (ug/ul) | DNA (ul) | DW (ul) |
|---|
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.
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:
Follow these steps to calculate the age of a mouse in Excel:
=TODAY()=B2-A2=INT(C2/7)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.
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.
"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.
To prevent Star Activity and optimize the use of restriction enzymes, consider the following approaches:
Conduct literature research on the specific restriction enzyme you plan to use to understand its recognition site and conditions for optimal activity.
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.
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.
Empirically monitor or prevent Star Activity by choosing the most suitable restriction enzyme from a variety of options.
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.
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.
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.
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.
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.
You can use this web app to set and run a simple timer. Follow these steps:
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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.
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
◈ 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.
◈ 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 (++)
◈ 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.
◈ 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.
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.
◈ 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.
◈ 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.
◈ 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.
◈ Measure the protein concentration of the extracted fractions (BCA assay).
◈ Use the fractions immediately or aliquot and freeze at -80°C for future use.
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.
Easily calculate the required volumes of DW, Master Mix, and Primers for your PCR experiments. This tool automatically adjusts for pipetting errors based on sample count.
Note: 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 may be adjusted based on objectives and sample types.
* Calculation base: PCR mixture 19 μl + cDNA 1 μl (Total 20 μl reaction)
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!"
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:
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.
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.
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.
Keep sample handling times as short as possible.
Avoid unnecessary freeze-thaw cycles.
Ensure that isopropanol used for RNA precipitation is stored at -20°C or colder.
Perform RNA precipitation steps at -20°C or colder.
When resuspending RNA pellets, vortex gently or pipette up and down gently to avoid shearing.
Check RNA quality and integrity using gel electrophoresis, Bioanalyzer, or similar methods.
Store RNA samples at -80°C for long-term storage.
Use RNase-free tubes and ensure proper sealing to prevent sample contamination.
Use gloves specifically designed for RNA work to minimize skin contact.
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!
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!
High-purity RNA is crucial, especially for DNA chip analysis where impurities (carbohydrates, proteins) can cause high background signals. Preventing genomic DNA contamination is also vital.
Assessing RNA purity before experiments is essential as it directly impacts results.
Denature 1-2 µg of total RNA (65°C, 10 min) and run on a 1% agarose gel.
Measure absorbance at 260nm (A260) and 280nm (A280).
Formula: Concentration = 40 µg/ml × A260 × Dilution Factor
(Assumption: A260=1 corresponds to 40 µg/ml RNA)
Scenario: 100 µl sample, A260 = 0.65, Dilution = 50x
1. Concentration = 40 × 0.65 × 50 = 1,300 µg/ml
2. Total Amount = 1,300 µg/ml × 0.1 ml = 130 µg
For the most accurate results, consider using automated detection devices (e.g., Bioanalyzer) alongside nucleic acid gel electrophoresis.
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.
: 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 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 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 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:
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.
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