DNA Dilution Calculator
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) |
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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."
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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.
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