Transformation-Competent E. coli preparation
DNA Ligation and Transformation Protocols
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1. Inoculate 1 ml from overnight culture into 100 ml Psi broth (scale up or down as needed). Incubate at 37 C with aeration to A550=0.48
2. Ice 15 min.
3. Pellet cells in appropriate centrifuge tube 3-5000 x g 5 min (~5000 rpm in a Sorvall SS-34 rotor)
4. Discard supernatant and add 0.4 volume (ie of original volume, here it is 40 ml) TfbI, resupend and ice 15 min.
5. Pellet cells as in #3.
6. Discard supernatant and resupend in 0.04 volume TfbII, ice 15 min and either use immediately or quick freeze at -70C for storage. I usually save these in 0.25 to 0.5 ml aliquots. Quick freeze in ethanol-dry ice or liquid nitrogen prior to storage in a -70 to -80 C freezer. Thaw on ice just before using in a transformation experiment.
I typically transform 50 ul cells with 2-10 ul of a ligation reaction, and you should get between 1x10exp8 to 1x10exp9 cfu's/ug DNA.
| compound | amount |
|---|---|
| Bacto yeast extract | 5 g |
| Bacto Tryptone | 20 g |
| magnesium sulfate | 5 g |
| pH 7.6 with potassium hydroxide |
| compound | amount | final molarity/conc. |
|---|---|---|
| potassium acetate | .588 g | 30 mM |
| rubidium chloride | 2.42 g | 100 mM |
| calcium chloride | 0.294 g | 10 mM |
| manganese chloride | 2.0 g | 50 mM |
| glycerol | 30 ml | 15% v/v |
| pH 5.8 with dilute acetic acid |
| compound | amount | final molarity/conc. |
|---|---|---|
| MOPS | 0.21 g | 10 mM |
| calcium chloride | 1.1 g | 75 mM |
| rubidium chloride | 0.121 g | 10 mM |
| glycerol | 15 ml | 15% v/v |
| pH 6.5 with dilute NaOH |
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1. Inoculate from an overnight grown in LB.
2. Grow in 250 ml "SOB" at 18C until OD600 = 0.6.
2. On ice for 10 minutes.
3. Spin at 2500 x g (5000 rpm in a Sorvall GSA or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
4. Resuspend cells gently in 80 ml of ice cold "TB". 5. On ice for 10 minutes.
6. Spin at 2500 x g (5000 rpm in a Sorvall GSA, 5500 rpm in a Sorvall SS-34, or 3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
7. Resuspend cells gently in 20 ml of ice cold "TB".
8. Add DMSO to a final concentration of 7%.
9. Place on ice for 10 minutes.
10. Aliquot into 1-2 ml and freeze in liquid nitrogen.
11. Store in liquid nitrogen.
| SOB | 2% (w/v) bacto tryptone | TB | |
|---|---|---|---|
| 0.5% (w/v) yeast extract | 10 mM Pipes | ||
| 10 mM NaCl | 55 mM MnCl2 | ||
| 2.5 mM KCl | 15 mM CaCl2 | ||
| 10 mM MgCl2 | 250 mM KCl | ||
| 10 mM MgSO4 | adjust pH to 6.7 with 5N KOH prior to adding the MnCl2 thanks to Markus Schneemann for the tip! | ||
| pH 6.7 - 7.0 |
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Cosmid Cloning: Cell preparation, DNA packaging, and Cell Transfection
Protocol taken from Stratagene's Gigapack packaging extracts instruction manual
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Tim Fitzwater, SomaLogic, Inc. 15-Aug-2002 (posted by permission, 8/16/02)
1. Filtration units should be prerinsed to remove Tween-20 present in the membranes as a wetting agent. Microfuge tubes should be siliconized and the use of aerosol resistant tips is strongly recommended.
2. 10x Potassium Glutamate Buffer (KGB)[1] consists of 1 M potassium glutamate, 0.25 M Tris acetate, pH 7.6, 0.1 M magnesium acetate, 5 mM b-mercaptoethanol and 500 μg/mL BSA. Combine 3.33 mL of 3 M potassium glutamate, 625 μL of 4 M Tris acetate, pH 7.6, 1 mL of 1 M magnesium acetate, 3.5 μL of 14.3 M b-mercaptoethanol, 500 μL of 10 mg/mL BSA and 4.54 mL of Type I water. Prepare 1 mL aliquots and store at -20°C. A salt/magnesium gradient will occasionally form if the tube is frozen. Mix well before use. A working tube may be kept at 4°C for over 1 year.
3. 5x Tris Acetate Ligation Buffer (TALB) is recommended for sticky end ligations and consists of 0.165 M Tris acetate, pH 7.6 at 25°C, 0.33 M potassium acetate, 50 mM magnesium acetate, 500 μg/mL BSA, 5 mM ATP, 0.05% NP-40 and 2.5 mM DTT.[2] The inclusion of NP-40 is useful for situations in which there are extremely limited amounts of DNA available, and can double the number of transformants in such cases by preventing DNA from adhering to the tube walls (data not shown). DTT is added as a separate component, as 100 mM magnesium tends to precipitate out when combined with 5 mM DTT. Combine 165 μL of 1 M Tris acetate, pH 7.6, 330 μL of 1 M potassium acetate, 50 μL of 1 M magnesium acetate, 50 μL of 10 mg/mL acetylated BSA, 50 μL of 1% NP-40, 50 μL of 100 mM ATP and 305 μL of Type I water. Aliquots are stable indefinitely at -20oC in the absence of DTT. Do not forget to add 1 μL of 100 mM DTT to each 20 μL ligation reaction. (Similar buffers are commercially available, without ATP or NP-40, as New England Biolabs Restriction Buffer 4, Fermentas Y/Tango, Roche Molecular Buffer A, etc.)
4. 1 M Tris acetate, pH 7.6: Method 1: Dissolve 18.12 g of Tris acetate in 80 mL of Type I water. Adjust the pH to 7.6 with NaOH and bring the volume to 100 mL with Type I water. Be sure that the pH probe is Tris-approved. Filter sterilize. Method 2: Dissolve 12.114 g of Tris base in 80 mL of Type I water. Adjust the pH to 7.6 with glacial acetic acid and bring the volume to 100 mL with Type I water. Filter sterilize. The optimum pH for T4 DNA ligase is 7.2-7.8. Tris buffer that is pH 7.6 at 22°C is pH 7.35 at 14°C.
5. 1 M potassium acetate: Dissolve 19.63 g of potassium acetate in 80 mL of Type I water. Adjust the volume to 100 mL and filter sterilize.
6. 1 M magnesium acetate: Dissolve 21.446 g of magnesium acetate in 80 mL of Type I water. Adjust the volume to 100 mL and filter sterilize.
7. 5x Blunt End Ligation Buffer (BRL Ligation Buffer):[3] consists of 250 mM Tris-HCl, pH 7.6, 50 mM MgCl2, 5 mM ATP, 5 mM DTT, 25% (w/v) PEG 8000. To prepare 10 mL of buffer, weigh 2.5 g of PEG 8000 in a 15 mL Falcon 2097 tube that has been treated with antistatic spray or wiped with a sheet of fabric softener. Microwave a 100 mL bottle of Type I water for 1 minute on High. Add 4.4 mL of hot water and 2.5 mL of room temperature 1 M Tris-HCl, pH 7.6 to the PEG and immediately mix to dissolve. Cool the mix to room temperature and add 500 μL of 1 M MgCl2, 500 μL of 100 mM ATP and 50 μL of 1 M DTT to a final volume of 10 mL. Aliquot for storage at -20°C. Failure to heat the water will result in PEG that will take approximately 24 hours to dissolve.