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    Competant Cells and Transformation

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    Swetha Ramesh
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    © All Rights Reserved
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    of 3
    Protocol 25 Preparation and Transformation of Competent E. coli using Calcium Chloride Tw. FOLLOWING SIMPLE AND RAPID VARIATION OF THE TECHNIQUE published by Cohen et al. (1972) is frequently used to prepare batches of competent bacteria that yield 3 x 10° to 2 x 10” trans. formed colonies/ug of supercoiled plasmid DNA. This efficiency of transformation is high enough to allow all routine cloning in plasmids to be performed with ease. Competent cells made by this procedure may be preserved at ~70°C, although there may be some deterioration in the efficiency of transformation during prolonged storage. MATERIALS _ Buffers and Solutions Please see Appendix 1 for components of stock sol Dilute stock solutions to the appropriate concentrat CaCI,2H,0 (7 9) ‘When preparing competent calls, thaw a 10-mlaiguot ofthe stock solution and dilute ito 100 ml with 90 mi of pure HO. Sterilz the solution by filtration through a prerinsed Nalgene fer (0.45-u1m pore sire), an then chill it 10 (FC. or Standard transformation buifer (TFB) (please see Protocol 23, Step 1) For many strains of E.coli, standard TFB (Hanahan 1983) may be used instead of CaCl, with equivalent tor better results, MgCl,-CaCl, solution, ice cold ions, buffers, and reagents. Media LB or SOB medium for initial growth of culture SOB agar plates comaining 20 m¥i MgSO, and the appropriate antibiotic Standard SOB contains 10 mM MgSO, SOC medium Approximately 1 ml of this medium i required for each transformation reaction, Nucleic Acids and Oligonucleotides Plasmid DNA (recombinant plasmid) ‘Construct using one of the methods described in Protocols 17 through 22 ofthis chapter 1116 Protocol 25: Preparation and Transformation of Competent E. coli using Calcium Chloride 16117 Centrifuges and Rotors Somall GSA rotor or equivalent Special Equipment Polypropylene tube (50 mb, chilled in ice Polypropylene tubes (17 x 100 mm; Falcon 2059), chilled in ice Water bath preset to 42°C METHOD A IMPORTANT All steps in this procedure should be carried out aseptically. Preparation of Cells 1. Pick a single bacterial colony (2-3 mm in diameter) from a plate that has been incubated for 16-20 hours at 37°C. Transfer the colony into 100 ml of LB broth or SOB medium ina |-liter flask. Incubate the culture for 3 hoursat 37#C with vigorous agitation, monitoring the growth of the culture. As a guideline, 1 ODgoy of a culture of E, coli strain DH1 contains ~10” bacte- riafml. For efficient transformation, it eset hat the numberof viable celle not exced 10 cell which for most strains of Ecos equvslent to an OD, of -04, To ensure thatthe culture does not grow t0a higher density, measre the ODja of the etre every 15-20 minutes, Plot a gph ofthe data so thatthe time when the OD, ofthe cure approaches an be predicted with Some accuracy Begin to harvest the culture hen the OD, teaches 0.35 Because the relationship between the OD yy and the numberof viable cells varies substantially from strain to strain the specrophotomte must be calibrate by measuring the OD, fa grow ing eultre ofthe particular stein of Eclat diferent times ints growth eye and determining ‘he number of ible clls at each of thesetimes by plating distin ofthe culture on LBagar pts intheabsence of antibiotics 2, Transfer the bacterial cells to sterile, disposable, ice-cold 50-ml polypropylene tubes. Cool the cultures to 0°C by storing the tubes on ice for 10 minutes. 3. Recover the cells by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 10 min- tutes at $C 4. Decant the medium from the cel pellets. Stand the tubes in an inverted position on a pad of paper towels for 1 minute to allow the last traces of media to drain away. 5. Resuspend each pellet by swirling or gentle vortexing in 30 ml of ice-cold MgCl,-CaCl, sol tion (80 mM MgCl, 20 mM CaCl). 6. Recover the cells by centrifugation at 2700g (4100 rpm in a Sorvall GSA rotor) for 10 min- utes at 4°C, 7. Decant the medium from the cell pellets. Stand the tubes in an inverted position on a pad of paper towels for I minute to allow the last traces of media to drain away: 8. Resuspend the pellet by swirling or gentle vortexing in 2 ml of ice-cold 0.1 M CaCl, (or TFB) for each 50 ml of original culture. 9. At this point, either use the cells directly for transformation as described in Steps 10 through 16 below or dispense into aliquots and freeze at 70°C {please see Protocol 23, Step 12). 1118 Chapter 1: Plasmids and Their Usefulness in Molecular Cloning Transformation For most strains of &.colf(except for MC1061}, TFB may be used at this stage instead of ‘suivant or beter results, the cells may be stored at 4°C in CaCl, solution for 24-48 hours (Dagert and Ehrlich 1979) The efficiency of transformation increases four- to sixfold during the fist 12-24 hous of storage and thereafter decreases tothe orginal level Include all of the appropriate positive and negative controls (please see the panel on BACTERIAL TRANSFORMATION in Protocol 23) 10. i. 12. 13, 14. 15. 16. ‘To transform the CaCl,-treated cells directly, transfer 200 ul of each suspension of competent cells to a sterile, chilled 17 x 100-mm polypropylene tube using a chilled micropipette tip. Add DNA (no more than 50 ng in a volume of 10 tl or less) to each tube. Mix the contents of the tubes by swirling gently. Store the tubes on ice for 30 minutes. ‘Transfer the tubes to a rack placed in a preheated 42°C circulating water bath, Store the tubes in the rack for exactly 90 seconds, Do not shake the tubes. Heat shock isa crucial step. Ii very important thatthe cells be raised to exactly the right tem> perature atthe correct rate Rapidly transfer the tubes to an ice bath, Allow the cells to chill for 1-2 minutes. Add 800 ul of SOC medium to each tube. Incubate the cultures for 45 minutes in a water bath set at 37°C to allow the bacteria to recover and to express the antibiotic resistance marker encoded by the plasmid The cells may be gent recovery period, y agitated (50 cycle'minute or less ina rotary shaker) at 376C: during the lf screening by a-complementation, proceed to Protocol 27 for plating. Transfer the appropriate volume (up to 200 ul per 90-mm plate) of transformed competent cells onto agar SOB medium containing 20 mM MgSO, and the appropriate antibiotic: ‘When selecting for resistance to tetracycline, the entire transformation mixture maybe speed on a single plate or plate in tp agar. this case, collect he bacteria by centrifuging fo 20 seconds 2 room temperature in a miseofe, and then gently resuspend the el pellet in 10 pl of SOC ‘medium by tapping the sides ofthe tbe. {A IMPORTANT Seize a ben ls rod by coping it ino ethanol and then in he fame of «Bunsen tue. When the od fas cook o room temperature, spread the transformed cells gery ove the surface ofthe agar plate. When selecting for resistance to ampicillin, transformed cel shouldbe plated at ove density 10" colonies per 90-mm plate),and the pats should not be incubated for more than 20 hours at 37%: “hc eneyme f-lactamas is sereed ino the medium from ampiiln-resistan transformants and

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