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.

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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.

[EasyTools] PCR Mixture Calculator

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.

Sample Count (N):

Repeat Count (n):

Select Primer Concentration: 10 pmol stock 100 pmol stock

Calculation Result:

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)

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!