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:

1. Enter your desired hours, minutes, and seconds in the input fields.
2. Click the "Set Timer" button to configure the timer.
3. The timer will start counting down until the specified time elapses.
4. 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."

hour
minute
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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.

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.

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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.

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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.

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.

BCA Protein Assay Protocol

Pierce™ BCA Protein Assay Protocol

Materials and Equipment:

• Pierce™ BCA Protein Assay Kit (A55864)
• Sample (protein sample)
• BSA (Bovine Serum Albumin for creating a standard curve)
• Microplate (96 wells)
• Multi-pipette (for dispensing samples and standards)
• Spectrophotometer or plate reader

Steps:

1. Add your protein samples to each well of the microplate. Add 25-200 μg of protein to each sample well.

2. Dilute the standard solution to various concentrations, typically 0, 7.8, 15.6, 31.25, 62.5, 125, 250, 500 μg/mL, etc.

3. Prepare the Pierce™ BCA Reagent mixture: Mix 50 parts of Pierce™ BCA Reagent Solution A with 1 part of Pierce™ BCA Reagent Solution B. For example, mix 50 μL of Solution A with 1 μL of Solution B to create the working Pierce™ BCA Reagent solution. (A:B=50:1)

4. For each standard concentration and sample, add the Pierce™ BCA Reagent and mix well. Incubate the plate at 37°C for 30 minutes.

5. After the incubation, measure the absorbance at 562 nm using a spectrophotometer or plate reader.

6. Create a standard curve using the absorbance values for each standard. Plot the data and perform linear regression analysis.

7. Calculate the protein concentrations of your samples using the standard curve and the absorbance values obtained.