Max Efficiency DH5α competent Cells
Cat. No.:18258-012 Size: 1 ml
Lot No.: KA7704 Store at –70℃ Do not store in liquid nitrogen
Description
Quality control: Max efficiency DH5αCompetent cells consitently yield>1*109 transformatnts/ug
PUC19 with nonsaturating amounts(50pg) of DNA. Saturating amounts of pUC19 (25ng) generate>1*106 ampicillin-resistant colonies in a 100ul reaction
Transformation procedure
A stock pUC19 solution (0.01ug/ml) is provided as a control to determine the transformation efficiency. To obtain maximum transformation efficiency, the experimental DNA must be free of phenol, ethanol, protein and detergents.
1. Prepare a dry ice/ethanol bath and maintain at –70℃
2. Remove competent cells from –70℃ freezer, thaw on wet ice. Place required number of 17×100mm polypropylene tubes on ice
3. Gently mix cells, then aliquot 100ul competent cells into chilled polypropylene tubes
4. refreeze any unused cells in the dry ice/ethanol bath for 5min before returning them to the –70℃ freeze. Do not use liquid nitrogen.
5. To determine transformation efficiency, add 5ul (50pg) control DNA to one tube conaining 100ul competent cells. Move the pipette through the cells whild duspensing. Gently tap tube to mix.
6. for DNA from ligation reactions, dilute the reactions 5-fold in 10mM Tris-HCl (pH 7.5) and 1mM Na2EDTA. Add 1ul of the dilution to the cells (1-10ng DNA), moving the pipette through the cells while dispensing. Gently tap tube to mix
7. incubate cells on ice for 30 min
8. Heat-shock cells 45s in a 42℃ water bath; do not shake
9. Place on ice for 2min
10. Add 0.9 ml of room temperture S.O.C Medium
11. Shake at 225 rpm (37℃) for 1 hour
12. Dilute the reaction containing the control plasmid DNA 1:100 with S.O.C Medium. Spread 100ul of this dilution on LB or YT plates with 100ug/ml ampicillin
13. Dilute experimental reactions as necessary and spread 100-200 ul of this dilution as descried in Step 12
14. Incubate overnight at 37℃
Growth of transformations for plasmid preparations
DH5α Competent cells which have been transformed with pUC-based plasmids should be grown at 37℃ overnight in TB(2). A 100 ml growth in a 500 ml baffled shake flask will yield approxiamately 1 mg of pUC19 DNA
S.O.C. Medium preparation (1)
For optimal performance with Max efficiency DH5α Competent cells, Life Technology recommends its premixed formulation of S.O.C Medium. To prepare the medium yourself, we recommend the following formulation:
To 97ml distilled H2O add 2g bactotryptone, 0.55g yeast extract, 1ml 1M NaCl and 1ml 1M KCl. Stir to dissolve, autoclave, and coll to 55c. add 1ml 2M Mg2+ (1M MgCl2, 1M MgSO4) and 1ml 2M glucose. Filter the complete medium through a 0.2um filter unit. Filter units should be pre-filtered with distilled H2O before use to remove any toxic material from the filter. The pH should be 7.0±0.1
TB MEDIUM PREPARATION (2)
To 900ml distilled H2O, add 12g tryptone, 24g yeast and 4ml glycerol. Stir to dissolve, autoclave, and cool room temperature. Add 2.3g KH2PO4 and 12.5g K2HPO4 to distilled water, to a final volume of 100ml. Stir to dissolve, autoclave, and cool to room termperature. Add the phosphate buffer solution to the media solution and mix thoroughly.
NOTES:
1 Falcon 2059 tubes or other similarly shaped 17*100mm polypropylene tubes are required for optimal transformation efficiency Microcentrifuge tubes (1.5ml) can be used but the transformation efficiency will be reduced 3- to 10- fold.
2 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.
3. 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
4 Transformation efficiencies will be approximately 10-fold lower for ligation of inserts to vectors than for an intact control plasmid such as pUC19. Ligation reactions should be diluted 5-fold prior to using the DNA in a transformtion. Only 1ul of this dilution should be used. A standard ligation reaction (20ul) normally contains 100-1000ng of DNA. Therefore, the addition of 1ul of diluted DNA will result in adding 1-10ng of ligated DNA to the cells. We have observed that the cells begin to satureate with 10-50ng of DNA. Also our data show that the 5-fold dilution of ligation mixtures results in more efficient transformation.
5 MAX efficiency DH5alfa can supports the replication of M13mp vectors. However, DH5alfa is F- and cannot supports plaque formation. Therefore, log phase DH5alfa, et al., cells must be added to the top agar which should contain Bluo-gal or X-gal, final concentration 50ug/ml, and IPTG, final concentration 1mM. The competent cells should be added to top agar after lawn cells, IPTG and Bluo-gal or X-gal have been added. Incubation at 37c for 1hour is not required after addition of S.O.C Medium.
