Protocols

Introduction

MultiShot StripWell TOP10 Chemically Competent E. coli

The MultiShot StripWell TOP10 Chemically Competent E. coli Kit is chemically competent TOP10 E. coli packaged in a 96-stripwell plate to simplify medium and high throughput bacterial transformation. The stripwell format allows you to use only the number of wells you need for your particular application. 

MultiShot FlexPlate TOP10 Chemically Competent Cells

The MultiShot FlexPlate TOP10 Chemically E. coli Kit is packaged in 96-well plate that is divisible into 12 x 8-well segments for high-throughput applications

MultiShot 96-Well Plate TOP10 Chemically Competent E. coli

The MultiShot TOP10 Chemically Competent E. coli Kit is chemically competent TOP10 E. coli packaged in five 96-well plates to simplify high throughput bacterial transformation. After addition of DNA, cells can be transformed by heat-shock at 42°C using a heat block or thermocycler.

Genotype of TOP10

F- mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacX74 recA1 araD139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG

Materials

Each MultiShot TOP10 Kit contains the following reagents.

ReagentCompositionAmount
SOC medium2% Tryptone; 0.5% Yeast Extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl2; 10 mM MgSO4; 20 mM glucose5 x 15 mL
TOP10 cells—Variable
pUC1910 pg/µl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 850 µL (500 pg)

Protocol—MultiShot StripWell Plate

Before starting

  • Prepare a container of ice large enough to chill the number of wells you will be using.
  • Bring the vial of S.O.C. to room temperature.
  • Pre-heat a water bath to 42°C.

Procedure

  1. Remove a MultiShot StripWell plate from the freezer and remove the number of wells you need. Return any unused wells to the freezer. Place the wells in the container of ice. Cells should thaw within 1 minute.
  2. Carefully remove the strip of caps from each set of 8 wells and keep them for further use.
  3. Use a multi-channel pipet to add 2-5 µl DNA (2 pg-20 ng) to the wells. Keep the volume around 2 µl for uniform results.
  4. After adding the DNA, cover the wells with the caps and incubate the cells and DNA on ice for 30 minutes.
  5. Transfer the wells to the water bath and heat-shock for 30 seconds at 42°C.
    Note: Be careful not to contaminate the cells.
  6. Transfer the wells back to the ice and allow the wells to cool for 1 minute.
  7. Remove the caps and add 250 µl S.O.C. to each well. Re-cap the wells tightly.
  8. Incubate the wells at 37°C for 1 hour with shaking (225 rpm). We turn the wells on their side to increase aeration and secure them to the shaker.
  9. Plate the appropriate volume from each well.

Plating Volumes and Expected Results

The table below describes the type of DNA, amount transformed into MultiShot StripWell chemically competent cells, the volume plated, and the number of colonies.
Note: We use pUC19 to qualify the kit. Transformation efficiency should be > 1 x 108 cfu/µg and yield 100–300 colonies per plate. Variability should be no more than 5-fold between wells.

DNATypeAmount transformedVolume platedNumber of colonies
pUC19Supercoiled5 pg plasmid10 µl100–300
pCR2.1-TOPO plus amplified 750 bp insertTOPO Cloning3.4 ng vector + insert25 µl100–300

Protocol—MultiShot FlexPlate TOP10 Chemically Competent E. coli

MultiShot TOP10 Chemically Competent E. coli Protocol

Protocol—MultiShot 96-Well Plate

Before starting

  • Chill a 96-well metal heating block (VWR, Cat. No. 13259-260) on ice until the block is cold.
  • Bring the vial of SOC to room temperature.
  • Pre-heat a heat block or thermocycler containing a 96-well metal block to 42°C.
    Note:     You can also use a water bath, but be careful not to contaminate the cells.
  • If you are using a thermocycler, program the machine to hold the temperature at 42°C.

Procedure

  1. Remove a MultiShot plate from the freezer and place it in the chilled metal block. Cells should thaw within 30 seconds.
  2. Carefully remove the aluminum foil seal.
  3. Use a multi-channel pipet to add 2 µL DNA (2 pg-20 ng) to the wells. Keep the volume around 2 µL for uniform results.
    Note:  Ligation reactions and TOPO Cloning reactions may need to be diluted with sterile water or TE buffer to prevent excess colonies.
  4. Cover the cells with the supplied plastic lid and incubate the cells and DNA in the chilled block for 20 minutes.
  5. Transfer the cell plate to either the pre-warmed heat block or thermocycler and heat-shock for 30 seconds at 42°C.
  6. Transfer the cell plate back to the chilled block and allow the plate to cool for 1 minute.
  7. Remove the plastic lid and add 90 µL SOC to each well.
  8. Cover and incubate the plate at 37°C for 1 hour. It is not necessary to shake the plate.
  9. Plate the appropriate volume from each well.

Plating volumes and expected results

The table below describes the type of DNA, amount transformed into MultiShot chemically competent cells, the volume plated, and the number of colonies.
Note:We use pUC19 to qualify the kit. Transformation efficiency should be > 1 x 108 cfu/µg and yield 100–300 colonies per plate. Variability should be no more than 5-fold between wells.

DNATypeAmount transformedVolume platedNumber of colonies
pUC19Supercoiled2.5 pg vector10 µL100–300
pCR 2.1 plus PCR-amplified 750 bp insertTA ligation200 pg vector + 32 pg insert (diluted 1:50 from original ligation reaction)Entire volume (~110 µL) 100–400
pCRXL-TOPO plus PCR-amplified 7 kb insertTOPO Cloning reaction~3 ng vector + 14 ng insert (undiluted)Entire volume (~110 µL) 100–200

LT082