Lactococcus lactis Electroporation Competent Cells

$295.00


    12x50μl Strain MG1363 - $295.00 (Cat.# 1291-24 )

    12x50μl Strain IL1403 - $295.00 (Cat.# 1292-24 )

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Description

Intact Genomics Electroporation Competent Lactococcus lactis Cells are customer strains optimized for the highest transformation efficiencies which is ideal for applications requiring high transformation efficiencies, such as with cDNA or gDNA library construction.

Specifications
Competent cell type:                Electrocompetent
Species:                                    L. lactis
Strain:                                      IL 1403  or  MG1363
Format:                                    Tubes
Transformation efficiency:         ≥ 1 x 106 cfu/µg pNZ8148 DNA
Blue/white screening:              No
Shipping condition:                  Dry ice

Reagents Needed for One Reaction
ElectroCompetent Lactococcus lactis Cells :          50 µl
DNA (or pNZ8148 Control, 50 ng/µl):                      1 µl
Recovery medium:                                                   1 ml

Storage
ElectroCompetent Lactococcus lactis Cells : -80 ºC
pNZ8148 control DNA:                                   -20 ºC
Recovery medium:                                          4 ºC

Quality Control
Transformation efficiency is tested by using the pNZ8148 control DNA supplied with the kit and using the protocol in this manual. Transformation efficiency should be ≥1 x 106 CFU/µg pNZ8148 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines
Follow these guidelines when using Electroporation Competent Lactococcus lactis Cells:

  • Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting.
  • Thaw competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by tapping the tube gently. Do not mix cells by pipetting or vortexing.

 Note: A high-voltage electroporation apparatus such as Bio-Rad Gene Pulser II #165-2105, capable of generating field strengths of 16 kV/cm is required.

 Calculation of Transformation Efficiency
Transformation Efficiency (TE) is defined as the number of colony forming units (cfu) produced by transforming 1µg of plasmid into a given volume of competent cells.

TE = Colonies/µg/Dilution

Transform 1 µl of (50 ng/µl) pNZ8148 control plasmid into 50 µl of cells, add 949 µl of Recovery Medium. Dilute 100 µl of this in 900 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate in two days. If you count 300 colonies, the TE is calculated as follows:
Colonies = 300
µg of DNA = 0.05
Dilution = 50/1000 x 100/1000 = 0.005
TE = 300/.05/.005 = 1.2×106

1291-24 1292-24

Additional information

μl

12×50μl Strain MG1363, 12×50μl Strain IL1403

Transformation Protocol

Use this procedure to transform Electroporation Competent Lactococcus lactis Cells. Do not use these cells for chemically transformation.

1)    Place sterile cuvettes and microcentrifuge tubes on ice.

2) Remove competent cells from the -80 °C freezer and thaw completely on wet ice (10-15 minutes).

3)    Aliquot 1 µl (1 ng -10 µlg) of DNA to the chilled microcentrifuge tubes on ice.

4) When the cells are thawed, add 50 μl of cells to each DNA tube on ice and mix gently by tapping 4-5 times. For the pNZ8148 control, add 1 µl of (50 ng/µl) DNA to the 50 µl of cells on ice. Mix well by tapping. Do not pipette up and down or vortex to mix, this can harm cells and decrease transformation efficiency.

5) Pipette 51 µl of the cell/DNA mixture into a chilled electroporation cuvette without introducing bubbles. Quickly flick the cuvette downward with your wrist to deposit the cells across the bottom of the well and then electroporate.

6) Immediately add 949 µl of Recovery Medium or any other medium of choice to the cuvette, pipette up and down three times to re-suspend the cells. Transfer the cells and Recovery Medium to an Eppendorf tube.

7) Seal the closed tube caps with parafilm and quickly warm tubes to 37 °C using a water bath.

8) Incubate tubes at 37 °C for 3 hours with no shaking.

9) Dilute the cells as appropriate then spread 20-200 μl cells onto a pre-warmed selective plate. For the pNZ8148 control, dilute the cells 1/10 and plate 50 μl of diluted transformants onto an MRS plate containing 10 μg/ml chloramphenicol. Use sterilized spreader or autoclaved ColiRoller™ plating beads to spread evenly.

10) Incubate the plates for 2 days at 37 °C under anaerobic conditions.

Electroportation settings
Mode Time constant protocol

Voltage (V) 2,000 V

Time constant (τ) 5 ms

Cuvette 1 mm

 

 

Lactococcus lactis Electroporation Competent Cells Manual