Protocols

Introduction

The OmniMAX 2 T1 Phage-Resistant (T1R) E. coli strain is an improved chemically competent cell line, perfect for use in all cloning applications, including the Gateway Technology. OmniMAX 2 -T1R cells offer the highest transformation efficiency of any chemically competent cells in a One Shot format with >5 x 109 transformants/µg pUC19. They also provide efficient transformation of highly methylated DNA, since OmniMAX 2 -T1Rcells lack the E. coli K12 restriction systems (mcrA (mrr hsdRMS-mcrBC)). In addition, the strain carries the tonA genotype, which confers resistance to T1 and T5 phage infection. This protects your samples and minimizes the possibility of downtime in your lab due to phage contamination. The highly versatile OmniMAX 2 -T1R strain offers the following benefits: 

  • Ideal for transformation of Gateway and TOPO reactions
  • Resistance to T1 and T5 phage (tonA)
  • Construction of more representative genomic libraries due to the elimination of mcrA, mcrBC, mrr, and hsdRMS
  • Blue/white screening of recombinant clones (lacZM15)


Genotype

F´ {proAB lacIq lacZM15 Tn10(TetR ) (ccdAB)} mcrA (mrr hsdRMS-mcrBC) 80(lacZ)M15 (lacZYA-argF)U169 endA1 recA1 supE44 thi-1 gyrA96 relA1 tonA panD

Materials

Each kit contains the reagents listed below. Transformation efficiency is greater than 5 x 109 cfu/µg DNA.

ItemCompositionAmount
S.O.C. Medium (store at room temperature or +4°C)2% Tryptone, 0.5% Yeast Extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose6 mL
OmniMAX 2-T1R Cells—20 x 50 µL
pUC19 Control DNA10 pg/µL in 5 mM Tris-HCl, 0.5 mM EDTA, pH 850 µL

Contents and storage

OmniMAX 2 T1 Phage-Resistant E. coli are supplied as chemically competent cells in a One Shot format. Store at –80°C

TOP

Transformation Procedure

General guidelines

Perform the following before starting the transformation procedure:

  1. Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting. Thaw One Shot competent cells on ice, and transform cells immediately following thawing. After adding DNA, mix by swirling or tapping the tube gently. Do not mix cells by pipetting.
  2. One Shot OmniMAX 2-T1R cells are lacIq and require IPTG to induce expression from the lac promoter. Spread 40 µL of 100 mM IPTG on top of the agar. Let the IPTG diffuse into the agar for approximately 1 hour. If blue/white screening is required to select for transformants, spread 40 µL of 40 µg/mL X-Gal in dimethylformamide in addition to IPTG on top of the agar. Let the X-Gal and IPTG diffuse into the agar for approximately 1 hour.

Before starting

Perform the following before starting the transformation procedure:

  • Equilibrate a water bath to 42°C.
  • Warm the vial of S.O.C. Medium (supplied with the kit) and LB Medium to room temperature.
  • Spread IPTG or IPTG and X-Gal onto LB agar plates containing antibiotic, if desired.
  • Warm the selective plates in a 37°C incubator for 30 minutes (use one plate for each transformation). If you are including the pUC19 control, make sure that you have one LB agar plate containing 100 µg/mL ampicillin.

Transformation procedure

Use the following procedure to transform One Shot OmniMAX 2-T1R chemically competent E. coli. We recommend including the pUC19 control plasmid DNA supplied with the kit in your transformation experiment to verify the efficiency of the competent cells. Do not use these cells for electroporation.

  1. Thaw, on ice, one vial of One Shot OmniMAX 2-T1R chemically competent cells for each transformation.
  2. Add 1 to 5 µL of the DNA (10 pg to 100 ng) into a vial of One Shot cells and mix gently. Do not mix by pipetting up and down. If you are transforming the pUC19 control, add 1 µL (10 pg) into a separate vial of One Shot cells and mix gently.
  3. Incubate the vial(s) on ice for 30 minutes.
  4. Heat-shock the cells for 30 seconds at 42°C without shaking.
  5. Remove the vial(s) from the 42°C bath and place them on ice for 2 minutes.
  6. Add 250 µL of pre-warmed S.O.C. Medium to each vial.
  7. Cap the vial(s) tightly and shake horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.
  8. Before plating, dilute the transformation mix 1:50 into LB Medium (e.g. remove 10 µL of the transformation mix and add to 490 µL of LB Medium).
  9. Spread 25–100 µL of the diluted transformation mix on a pre-warmed selective plate. Store the remaining undiluted and diluted transformation mix at +4°C. Additional cells may be plated out the next day, if desired.
  10. Invert the plate(s) and incubate at 37°C overnight.
  11. Select colonies and analyze by plasmid isolation, PCR, or sequencing.

Calculating transformation efficiency

Use the following formula to calculate the transformation efficiency as transformants (in cfu) per µg of plasmid DNA, where DF is the dilution factor.

# of colonies
10 pg transformed DNA		×106 pg
µg		× 300 µL total transformation volume
X µL plated		×50 (DF)=# transformants
µg plasmid DNA

References

  1. Blumenthal, R. M. (1989). Cloning and Restriction of Methylated DNA in Escherichia coli. Focus 11, 41-46.
  2. Grant, S. G., Jessee, J., Bloom, F. R., and Hanahan, D. (1990). Differential Plasmid Rescue from Transgenic Mouse DNAs into Escherichia coli Methylation-restriction Mutants. Proc. Natl. Acad. Sci. USA 87, 4645-4649.
C4040.pps      23-8-2006