F- φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-, mk+) phoA supE44 λ-thi-1 gyrA96 relA1 tonA
|Component||Amount per Vial|
|DH5α T1 Phage Resistant Competent Cells||200 μl|
|pUC19 DNA (0.01 μg/ml)||100 μl|
MAX Efficiency DH5α T1 Phage Resistant Competent Cells consistently yield > 1.0 x 10 9 transformants/μg pUC19 with non-saturating amounts (50 pg) of DNA. Saturating amounts of pUC19 (25 ng) generate > 1 x 10 6 ampicillin-resistant colonies in a 100-μl reaction. The cells are tested to confirm resistance to the bacteriophage T5, which is a standard test for cross-resistance to bacteriophage T1 (1).
- Thaw competent cells on wet ice. Place required number of 17 x 100 mm polypropylene tubes (Falcon® 2059; see Note 1) on wet ice.
- Gently mix cells, then aliquot 100 μl competent cells into chilled polypropylene tubes.
- Refreeze any unused cells in the dry ice/ethanol bath for 5 minutes before returning them to the -70°C freezer. Do not use liquid nitrogen.
- To determine transformation efficiency, add 5 μl (50 pg) control DNA to one tube containing 100 μl competent cells. Move the pipette through the cells while dispensing. Gently tap tube to mix.
- For DNA from ligation reactions, dilute the reactions 5-fold in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA. Add 1 μl of the dilution to the cells (1-10 ng DNA), moving the pipette through the cells while dispensing. Gently tap tubes to mix.
- Incubate cells on ice for 30 minutes.
- Heat-shock cells 45 seconds in a 42°C water bath; do not shake.
- Place on ice for 2 minutes.
- Add 0.9 ml of room temperature S.O.C. Medium (Cat. No. 15544-034).
- Shake at 225 rpm (37°C) for 1 hour.
- Dilute the reaction containing the control plasmid DNA 1:10 with S.O.C. Medium. Spread 100 μl of this dilution on LB or YT plates with 100 μg/ml ampicillin and 50 μg/ml X-gal (Cat. No. 15520-034) or Bluo-gal.
- Dilute experimental reactions as necessary and spread 100-200 μl of this dilution as described in Step 11.
- Incubate overnight at 37°C.
DH5α T1 Phage Resistant Competent Cells which have been transformed with pUC-based plasmids should be grown at 37°C overnight in TB(3). A 100-ml growth in a 500-ml baffled shake flask will yield approximately 1 mg of pUC19 DNA.
- Falcon® 2059 tubes or other similarly shaped 17 × 100 mm polypropylene tubes are required for optimal transformation efficiency. Microcentrifuge tubes (1.5 ml) can be used but the transformation efficiency will be reduced 3- to 10-fold.
- For best results, each vial of cells should be thawed only once. Although the cells are refreezable, subsequent freeze-thaw cycles will lower transformation frequencies by approximately 2-fold.
- Media other than S.O.C. Medium can be used but the transformation efficiency will be reduced. Expression in Luria Broth reduces transformation efficiency a minimum of 2- to 3-fold.Cat. No. 12034-013
- MAX Efficiency DH5α T1 Phage Resistant Competent Cells can support the replication of M13mp vectors. However, DH5α T1 is F- and cannot support plaque formation. Therefore, log phase DH5α-FT, DH5αF', DH5αF'IQ, JM101 or JM107 cells must be added to the top agar which should contain X-gal (Cat. No. 15520-034) or Bluo-gal, final concentration 50 μg/ml, and IPTG (Cat. No. 15529-019), final concentration 1 mM. The competent cells should be added to top agar after lawn cells, IPTG and Bluo-gal or X-gal have been added. Incubation at 37°C for 1 hour is not required after addition of S.O.C. Medium.
- Transformation efficiency (CFU/μg):
CFU in control plate × 1 × 106 pg × dilution factor(s)
pg pUC19 used in transformation μg
For example, if 50 pg pUC19 yields 100 colonies when 100 μl of a 1:100 dilution is plated, then:
CFU/μg = 100 CFU × 1 × 106pg × 1 ml × 102 = 2 × 109
50 pg μg 0.1 ml plated
- Killman, M. (1996) J. Bact. 178,6913.
- Hanahan, D. (1983) J. Mol. Biol. 166, 557.
- Tartof, K. D. and Hobbs, C. A. (1987) Focus® 9:2, 12.