Routine Cloning Using Top10 Competent Cells

One Shot TOP10 E. coli are provided at a transformation efficiency of 1 x 10⁹ cfu/µg supercoiled DNA and are ideal for high-efficiency cloning and plasmid propagation. They allow stable replication of high-copy number plasmids. The genotype of TOP10 Cells is similar to the DH10B strain, and offers the following features: 

• hsdR for efficient transformation of unmethylated DNA from PCR amplifications
• mcrA for efficient transformation of methylated DNA from genomic preparations
• lacZM15 for blue/white color screening of recombinant clones  
• endA1 for cleaner preparations of DNA and better results in downstream applications due to the elimination of non-specific digestion by Endonuclease I  
• recA1 for reduced occurrence of non-specific recombination in cloned DNA

Genotype:
F- mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 ara139 (ara-leu)7697 galU galK rpsL (Str^R) endA1 nupG>

Each kit contains the following:

All kits contain the following reagents:

• 50 ml supercoiled pUC19 plasmid (10 pg/ml in 5 mM Tris-HCl, 5 mM EDTA, pH 8) for testing efficiency
• S.O.C Medium (6 ml) for plating

Introduction

This section provides two procedures to transform One Shot TOP10 chemically competent E. coli. See Selecting a One Shot Chemical Transformation Procedure below to help you choose the best procedure to use for your needs.
 
Selecting a One Shot Chemical Transformation Procedure

Two procedures are provided to transform One Shot TOP10 chemically competent E. coli. Consider the following factors when choosing which procedure to use. Note that if you use the rapid chemical transformation procedure, fewer transformants will be obtained.

The rapid chemical transformation procedure is only suitable for transformations using ampicillin selection. If you will be using any other antibiotic for selection (e.g. kanamycin), use the regular chemical transformation procedure.
 
 
Materials Supplied by the User
You will need the following items for transformation:

• 37°C shaking and non-shaking incubator
• 10 cm diameter LB agar plates with appropriate antibiotic
• Ice bucket with ice
• 42°C water bath

 
Before Starting

• Equilibrate a water bath to 42°C.
• Warm the vial of S.O.C medium to room temperature.
• Spread X-Gal onto LB agar plates with antibiotic, if desired for blue/white selection.
• Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation). 

Important: It is essential that LB plates containing 100 mg/ml ampicillin are pre-warmed if you are performing the rapid chemical transformation procedure.
 
Chemical Transformation Procedure

The instructions provided below are for general use. Specific instructions for particular applications such as Zero Blunt PCR Cloning are provided in the manual for that kit. 

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 to 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 µl to 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.

 
Rapid Chemical Transformation Procedure

An alternative procedure is provided below for rapid transformation of One Shot chemically competent E. coli. This procedure is only recommended for transformations using ampicillin selection. Note: It is essential that selective plates be pre-warmed prior to spreading.

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 to 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 5 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. Incubate the vial(s) on ice for 5 minutes.
9. Spread 50 µl of cells on a pre-warmed, labeled LB agar plate containing 100 µg/ml ampicillin and incubate at 37°C overnight.
10. Select colonies and analyze by plasmid isolation, PCR, or sequencing.

Materials Supplied by the User

You will need the following items for transformation:

• 37°C shaking and non-shaking incubator
• 10 cm diameter LB agar plates with appropriate antibiotic
• Ice bucket with ice
• Electroporator
• Cuvettes (0.1 or 0.2 cm, see Note)
• 15 ml snap-cap tubes (one for each transformation)

 
One Shot Electrocomp cells are supplied in 50 µl single-use aliquots. Please refer to the user manual included with your electroporator for cuvette size and reaction volume. You may dispose of any unused cells.

Preparation

For each transformation, you will need one vial of competent cells and at least one selective plate.

• Thaw the vial of S.O.C medium and bring to room temperature.
• Spread X-Gal onto LB agar plates with antibiotic, if desired.
• Warm selective plates at 37°C for 30 minutes.
• Place cuvettes on ice.
• Thaw on ice 1 vial of One Shot Electrocomp cells for each transformation.

Electroporation Procedure

The instructions provided below are for general use. Specific instructions for particular applications such as TOPOXL PCR Cloning are provided in the manual for that kit.
 
Note:  For transformation of large plasmids, electroporation is preferred over chemical transformation because not only is the transformation efficiency higher, it is less biased against large recombinant plasmids.
 
Important: To avoid arcing, use only Electrocomp cells for electroporation. 

1. Set up your electroporator for bacterial transformation. Follow the manufacturer's instructions. 
2. Add 1-2 µl of each ligation reaction to the volume of cells recommended by the manufacturer (may be less than 50 µl). Mix gently with pipette tip. Do not mix by pipetting up and down. 
3. Transfer the cells to the chilled electroporation cuvette on ice. 
4. Electroporate the cells as per the manufacturer's recommended protocol. 
5. Quickly add 250 µl room temperature S.O.C medium and mix gently. 
6. Transfer the solution to a 15 ml snap-cap tube (i.e. Falcon) and shake for at least 1 hour at 37°C to allow expression of the antibiotic resistance gene. 
7. Spread 10 to 150 µl from each transformation on a prewarmed LB plate containing the appropriate antibiotic. The remaining transformation mix may be stored at +4°C and plated out the next day, if desired. 
8. Incubate the plates overnight at 37°C. 
9. Select colonies and analyze by plasmid isolation, PCR, or sequencing.

We recommend that you test the efficiency of the competent cells contained in the One Shot Kit. This can be accomplished by using the supercoiled pUC19 plasmid supplied with the kit as described below. 

  1.   Prepare LB agar plates containing 100 µg/ml ampicillin 

  2.   Transform 1 µl (10 pg) into 50 µl of competent cells according to the transformation protocol appropriate for the type of cells. 

  3.   Plate the control transformation as follows: 

*Just before plating the Electrocomp transformation mix, dilute 10 µl of the transformation mix with 90 µl of S.O.C medium. 

  4.   Incubate overnight at 37°C and count colonies. Calculate transformation efficiency using the formula below.
 
Calculation

Calculate the transformation efficiency as transformants per 1 µg of plasmid DNA.
 
For chemically competent cells, use the formula below to calculate transformation efficiency:

For Electrocomp cells, use the formula below to calculate transformation efficiency:

*Volume dependent on the volume of cells used and the amount of S.O.C. added.

Expected transformation efficiency: