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TOP10 chemically competent cells: Difference between revisions
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This protocol is a variant of the Hanahan protocol <cite>Hanahan91</cite> using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [[Media:pat6855494.pdf | Bloom05 patent]] as well. This protocol has been tested on TOP10, MachI and [[Talk:TOP10 chemically competent cells|BL21(DE3)]] cells. See [[Bacterial Transformation]] for a more general discussion of other techniques. The [[Media:pat6960464.pdf | Jesse '464 patent]] describes using this buffer for DH5α cells. The [[Media:pat6709852.pdf | Bloom04]] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. | This protocol is a variant of the Hanahan protocol <cite>Hanahan91</cite> using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the [[Media:pat6855494.pdf | Bloom05 patent]] as well. This protocol has been tested on TOP10, MachI and [[Talk:TOP10 chemically competent cells|BL21(DE3)]] cells. See [[Bacterial Transformation]] for a more general discussion of other techniques. The [[Media:pat6960464.pdf | Jesse '464 patent]] describes using this buffer for DH5α cells. The [[Media:pat6709852.pdf | Bloom04]] patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. | ||
==Materials== | |||
*Detergent-free, sterile glassware and plasticware (see procedure) | |||
*Table-top OD600nm spectrophotometer | |||
*[[SOB]] | |||
===CCMB80 buffer=== | |||
* 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L) | |||
* 80 mM CaCl<sub>2</sub>.2H<sub>2</sub>O (11.8 g/L) | |||
* 20 mM MnCl<sub>2</sub>.4H<sub>2</sub>O (4.0 g/L) | |||
* 10 mM MgCl<sub>2</sub>.6H<sub>2</sub>O (2.0 g/L) | |||
* 10% glycerol (100 ml/L) | |||
* adjust pH DOWN to 6.4 with 0.1N HCl if necessary | |||
** adjusting pH up will precipitate manganese dioxide from Mn containing solutions. | |||
* sterile filter and store at 4°C | |||
* slight dark precipitate appears not to affect its function | |||
==Procedure== | |||
===Preparing glassware and media=== | ===Preparing glassware and media=== | ||
Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic | Detergent is a major inhibitor of competent cell growth and transformation. Glass and plastic | ||
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** Good cells should yield around 100 - 400 colonies | ** Good cells should yield around 100 - 400 colonies | ||
* Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. | * Thawing and refreezing partially used cell aliquots dramatically reduces transformation efficiency by about 3x the first time, and about 6x total after several freeze/thaw cycles. | ||
===Measurement of competence=== | ===Measurement of competence=== | ||
