Intact Genomics (ig®) HB101 electrocompetent E. coli cells are suitable for high-efficiency transformation in a wide variety of applications such as cloning and sub-cloning. E. coli HB101 is a K12 x B hybrid strain, containing the recA13 mutation that minimizes recombination and helps insert stability. In addition, it carries the hsdS20(rB-mB-) restriction minus genotype which prevents cleavage of cloned DNA by endogenous restriction enzymes. HB101 strain does not support Alpha-complementation for blue/white screening.
Competent cell type: ElectroCompetent
Derivative of: HB101
Species: E. coli
Transformation efficiency: ≥ 4 x 1010 cfu/µg pUC19 DNA
Blue/white screening: Yes
Shipping condition: Dry ice
Reagents Needed for One Reaction
ig® HB101 electrocompetent cells: 25 µl
DNA (or pUC19 Control, 10 pg/µl): 1 µl
Recovery medium: 1 ml
ig® HB101 electroCompetent cells: -80 ºC
pUC19 control DNA: -20 ºC
Recovery medium: 4 ºC
F- Lambda- araC14 leuB6(Am) DE(gpt-proA)62 lacY1 glnX44(AS) galK2(Oc) recA13 rpsL20(strR) xylA5 mtl-1 thiE1 hsdS20(rB-, mB-)
Transformation efficiency is tested by using the pUC19 control DNA supplied with the kit and using the protocol given below. Transformation efficiency should be ≥4 x 1010 CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.
Follow these guidelines when using ig® HB101 ElectroCompetent 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.
Example 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 (10 pg/µl) pUC19 control plasmid into 50 µl of cells, add 950 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 100 colonies, the TE is calculated as follows:
Colonies = 100
µg of DNA = 0.00001
Dilution = 50/1000 x 10/1000 = 0.0005
TE = 100/.00001/.0005 = 2.0×1011271-12 1271-48