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Mice Anesthetization Protocols

Protocols

Dr AF Saeed

-

2nd October 2023

Mice Anesthetization Protocols

Anesthetizing mice is a common practice in biomedical research for various purposes, including surgery, imaging studies, or the collection of tissues. Several methods are available, including injectable and inhalant anesthetics.

1. Injectable Anesthetics: Some commonly used injectable anesthetics are ketamine, xylazine, and acepromazine.

– The combination of Ketamine (an analgesic) and Xylazine (a sedative) is commonly used for anesthesia in rodents.

– This mixture provides both anesthesia and analgesia, and the effects can last from 30 to 120 minutes, depending on the route of administration, the dose, and the specific animal.

– The typical dose is 60–100 mg/kg ketamine and 5–10 mg/kg xylazine by intraperitoneal (IP) injection.

Usage Example:

Mice were anesthetized via intraperitoneal injection of ketamine (100 mg per kg body weight) and xylazine (10 mg per kg body weight). (Yin et al., 2023, Nat. Immunol.)

2. Inhalant Anesthetics: Isoflurane is a commonly used inhalant anesthetic. Other options include Sevoflurane and Enflurane.

– Isoflurane is frequently the anesthetic of choice for minor surgeries due to its rapid induction and recovery times.

– It’s administered through a precision vaporizer. The general concentration for induction ranges from 2–5%, and for maintenance, 1–3%.

– Mice are generally placed in an induction chamber, and then the anesthesia is delivered through the nose.

3. Medetomidine-Midazolam-Fentanyl (MMF) Combination: In some instances, another combination that might be used is MMF.

– This combination is typically administered via the subcutaneous route.

– It provides excellent anesthesia and analgesia and lasts approximately 25–40 minutes, depending on the rodent and the dose.

Regardless of the method, all anesthetization techniques should be carefully monitored, ensuring the depth of anesthesia is sufficient but not excessive. Respiration rate, heart rate, and reflex activity can provide useful information about anesthesia depth. In the case of prolonged procedures, heat support should be supplied, as small animals such as mice can quickly become hypothermic.

After the procedure, mice must be monitored until they fully recover.

It’s crucial to remember that your institution’s animal care and use committee must approve every procedure involving anesthesia and mice.

All protocols should prioritize the animal’s well-being, particularly minimizing distress and pain. Please adhere to the “three-R” framework for the ethical use of animals in scientific research: Replacement, Reduction, and Refinement.

Please consult with a veterinarian or a professional technician to administer these procedures.

SDS-PAGE Gel Preparation

Protocols

Dr AF Saeed

-

2nd October 2023

1

SDS-PAGE Gel Preparation

1. Prepare the separation gel (10%). Mix in the following order:

H₂O	4.1 mL
Acrylamide/bis (30% 37.5:1; Bio-Rad)	3.3 mL
Tris-HCl (1.5 M, pH 8.8)	2.5 mL
SDS, 10%	100 µL
N,N,N′,N′-tetramethylethylene-diamine (TEMED) (Bio-Rad)	10 µL
Ammonium persulfate (APS), 10%	32 µL

After adding TEMED and APS to the SDS-PAGE separation gel solution, the gel will polymerize quickly, so add these two reagents when ready to pour.

2. Pour gel, leaving ∼2 cm below the bottom of the comb for the stacking gel. Make sure to remove the bubbles.

3. Layer the top of the gel with isopropanol. This will help remove bubbles at the top of the gel and keep the polymerized gel from drying out.

In about 30 minutes, the gel should be completely polymerized.

4. Remove the isopropanol and wash out the remaining traces of isopropanol with distilled water.

5. Prepare the stacking gel (4%). Mix in the following order:

H₂O	6.1 mL
Acrylamide/bis (30%, 37.5:1)	1.3 mL
Tris–HCl (0.5 M, pH 6.8)	2.5 mL
SDS, 10%	100 µL
TEMED	10 µL
Ammonium persulfate (APS), 10%	100 µL

6. Pour stacking gel on top of the separation gel.

7. Add combs to make wells. In ∼30 to 30 minutes, the stacking gel should become completely polymerized.

8. Clamp gel into the apparatus and fill both buffer chambers with gel-running buffer according to the instructions for the specific apparatus.

9. Load samples and molecular mass protein markers into wells for separation by electrophoresis.

Read More: SDS-Gel Electrophoresis Protocol

Tamoxifen Preparation and Treatment in Mice for Genetic Recombinations

Protocols

Dr AF Saeed

-

2nd October 2023

0

Tamoxifen Preparation and Treatment in Mice for Genetic Recombinations

Rationale: Tamoxifen is used to kick-start genetic recombinations in genetically engineered mouse models (GEMMs) and to activate genes through a cascade of events.

Here is a detailed step-by-step guideline on how to prepare and inject Tamoxifen into mice. Please ensure you are properly trained and authorized to handle laboratory mice before proceeding. Always follow animal welfare laws and your facility’s study protocol.

Tamoxifen Preparation

1. Calculate the appropriate concentration of tamoxifen for your experiment; the dose usually falls in the range of 40–100 mg/kg/day, but adjust according to the specifics of your study.

2. Tamoxifen is soluble in ethanol at 50 mg/mL. To create this stock solution, dissolve the required amount of tamoxifen in 100% ethanol.

3. After completely dissolved, dilute the stock solution with corn or sunflower oil to reach the intended final concentration.

4. Store it at -20 degrees Celsius for long-term storage.

Intraperitoneal Injection

1. Allow the tamoxifen solution to reach room temperature before the injection or warm at 37⁰C (recommended) before use to match the internal body temperature of mice for improved function.

2. Load a sterile insulin syringe (1 mL) with the appropriate volume to achieve the desired dosage.

3. For intraperitoneal (IP) injection, the mouse is restrained manually or using a suitable restrainer. Applying gentle pressure on both sides of the lower part of the abdomen makes the injection easier and more accurate.

4. Hold the syringe and needle like a dart and puncture the skin and peritoneum on the lower right side of the abdomen, avoiding vital organs.

5. Once inserted, depress the syringe slowly to inject the solution. Do not force it if you meet resistance.

6. Properly manage the needle after injection to prevent injuries and infections.

7. Monitor mice after the injection and record any changes in their behavior, weight, or appearance.

Be aware that tamoxifen administration can induce some side effects in mice, such as weight loss or pyometra in females; monitor them regularly.

Usage Example:

At 6 weeks of age, mice were intraperitoneally injected with 75 mg per kg (body weight) of tamoxifen (Sigma-Aldrich, T5648-5G) dissolved in corn oil once a day for 5 consecutive days (Yin et al., 2023, Nat. Immunol.).

This procedure must be done accurately and carefully. Future adjustments must be made based on the observed results. Always follow your Institutional Animal Care and Use Committee regulations and guidelines. If any issues occur, consult with your attending veterinarian.

Remember, this guide does not replace professional training, and an experienced investigator or veterinarian should supervise all steps until proficiency is achieved.

DNA Quantification using Qubit Fluorometer

Protocols

Dr AF Saeed

-

30th September 2023

1

DNA Quantification using Qubit Fluorometer

The Qubit Fluorometer is a device from Thermo Fisher Scientific used to quantify DNA, RNA, and protein in samples by using a fluorescent dye that binds to these molecules. The Qubit uses specialized assay kits, like the Qubit dsDNA HS (High Sensitivity) Assay Kit, for quantifying double-stranded DNA (dsDNA).

Here is a step-by-step process of using the Qubit fluorometer for DNA quantification:

Materials Required: Qubit fluorometer, Qubit dsDNA HS assay kit (or similar), Qubit assay tubes, Pipettes and tips, and DNA samples.

1. Prepare Qubit working solution: The working solution is made by diluting the Qubit dsDNA HS Reagent in the Qubit dsDNA HS Buffer. The standard dilution is 1 part reagent to 199 parts buffer. Mix the reagent before use as it may be prone to forming precipitates.

2. Prepare DNA Standards: The Qubit assay kit includes two DNA standards (Standard #1 and #2). These standards are used to calibrate the fluorometer. Add 190 µL of the working solution to each standard tube and then add 10 µL of the respective standard. Mix gently, then read the standards on the Qubit fluorometer.

3. Prepare DNA samples: Add 198 µL of the working solution into an assay tube for each DNA sample. Then, add 2 µL of the DNA sample. Mix the solution gently.

4. Measure DNA Standards and Samples: Place the tube containing Standard #1 into the Qubit and close the lid. On the screen of the Qubit, select “DNA” then “High Sensitivity” (or the appropriate assay) and tap “Read”. The fluorometer will read the standard and then prompt you to insert Standard #2. Then, proceed to read your samples.

5. Interpret the results: Once your sample is read, the Qubit will display the DNA concentration in your sample on the screen. The Qubit fluorometer software also allows you to export your data for further analysis.

Remember to follow good lab practices to prevent contamination of your samples. In addition, to maintain the stability of the fluorescent dye reagent, avoid extended periods of light exposure.

DNA Quantification using Nanodrop spectrophotometer

Protocols

Dr AF Saeed

-

30th September 2023

1

DNA Quantification using Nanodrop spectrophotometer

The nanodrop spectrophotometer is commonly used to quantify DNA concentration and assess purity. Here is a basic outline of the process:

1. Calibrate the machine: It’s usually done by using a blank solution that the machine will use as a base reading. This is typically the same solution your DNA is suspended in, such as pure distilled water or TE buffer.

2. Measure the Sample: Once the Nanodrop is calibrated, you can measure your sample. Apply a small amount (1-2 µl) of your sample to the measurement pedestal.

3. Take the Reading: Close the arm of the Nanodrop and take the reading. The machine will provide you with measurements at various wavelengths that can be used to quantify and qualify your sample.

The key measurements you’ll find in the Nanodrop report are:

– 260 nm: This is the absorbance value of your DNA. The concentration of your DNA (ng/µl) is calculated based on this value.

– 260/280 ratio: This ratio gives you an indication of the purity of your DNA. Pure DNA generally has a ratio of around 1.8, while lower ratios may indicate protein contamination.

– 260/230 ratio: This is another measure of purity. The optimal value tends to be above 2.0. A lower ratio could indicate organics, salts, or other compounds contamination.

Remember, while Nanodrop is a fast and easy method for quantifying DNA, it might not be the most accurate when it comes to very low concentrations or when the sample is contaminated. In such cases, fluorescence-based quantification methods like Qubit might be more appropriate.

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Mice Anesthetization Protocols

SDS-PAGE Gel Preparation

Tamoxifen Preparation and Treatment in Mice for Genetic Recombinations

DNA Quantification using Qubit Fluorometer

DNA Quantification using Nanodrop spectrophotometer

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