Introduction

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.
  
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.
 
Genotype of TOP10

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

Materials

Each MultiShot™ TOP10 Kit contains the following reagents. Each kit contains five 96-well plates of frozen competent cells.

Reagent CompositionAmount
 SOC medium
2% Tryptone; 0.5% Yeast Extract; 10 mM NaCl; 2.5 mM KCl; 10 mM MgCl2; 10 mM MgSO4; 20 mM glucose
5 x 15 ml
TOP10 cells---15 µl per well
pUC19 
10 pg/µl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8
50 µl (500 pg)

 

Each MultiShot™ StripWell Kit contains the following reagents. Fifty microliters of chemically competent TOP10 E. coli are supplied per well with a transformation efficiency of > 1 x 108 cfu/µg pUC19 DNA.

TOP

Transformation Protocol-MultiShot™ 96-Well Plate

Before Starting

  • Chill a 96-well metal heating block (VWR, Catalog 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.


DNA
Type
Amount Transformed
Volume Plated
Number of Colonies
pUC19
Supercoiled
2.5 pg vector

10 µl
100-300
pCR®2.1 plus PCR-amplified 750 bp insert
TA ligation
200 pg vector + 32 pg insert
(diluted 1:50 from original ligation reaction)
Entire volume (~110 µl) 100-400

pCR®XL-TOPO® plus PCR-amplified 7 kb insert

TOPO® Cloning reaction
~3 ng vector + 14 ng insert
(undiluted)
Entire volume (~110 µl) 100-200


TOP

Transformation 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.

DNA
Type
Amount Transformed
Volume Plated
Number of Colonies
pUC19
Supercoiled
5 pg plasmid
10 µl
100-300
pCR®2.1-TOPO® plus amplified 750 bp insert
TOPO® Cloning
3.4 ng vector + insert
25 µl
100-300


TOP
LT082