igMax DH10B ElectroCompetent Cells
Intact Genomics igMaxTM DH10B derivative ElectroCompetent cells are suitable for demanding cloning situations, such as synthetic bio-applications, BAC cloning, assembling large and multi-DNA fragments, and large construct transformation or cloning difficult targets, requiring the greatest number of transformants possible. Utilizing proprietary manufacturing methods, these cells allow for effective transformation of all large DNA molecules (≥10kb up to 350kb!).
Intact Genomics igMaxTM DH10B cells vs. Competitors DH10B Derivative transformation efficiency
Extremely large DNA molecules can effectively be transformed at a rate much higher than ANY competitor, as shown in the figure above.
Competent cell type: ElectroCompetent
Derivative of: DH10B
Species: E. coli
Transformation efficiency: ≥ 5 x 1010 cfu/µg pUC19 DNA
Blue/white screening: Yes
Shipping condition: Dry ice
Reagents Needed for One Reaction
igMaxTM DH10B ElectroCompetent cells: 25 µl
DNA (or pUC19 Control, 10 pg/µl): 1 µl
Recovery medium: 1 ml
igMaxTM DH10B ElectroCompetent cells:-80 ºC
pUC19 control DNA: -20 ºC
Recovery medium: 4 ºC
Intact Genomics igMaxTM ElectroCompetent DH10B cells have the following features:
- ≥5 x 1010cfu/µg efficiency with electroporation.
- 5~10×107 for 100~150 kb large DNA
F – mcrA ∆(mrr-hsdRMS-mcrBC) endA1 recA1 φ80dlacZ∆M15 ∆lacX74 araD139 ∆(ara, leu)7697 galU galK rpsL (StrR) nupG λ-
Transformation efficiency is tested by using pUC19, ~50kb, and >100kb plasmids. The pUC19 control DNA is supplied with the kit and using the protocol given below. Transformation efficiency should be ≥5 x 1010 CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.
Follow these guidelines when using Intact Genomics igMaxTM DH10B 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.
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 25 µl of cells, add 975 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 250 colonies, the TE is calculated as follows:
Colonies = 250
µg of DNA = 0.00001
Dilution = 25/1000 x 10/1000 = 0.00025
TE = 250/.00001/.00025 = 10.0×10101284 1284-24