Protocols
In vivo
Escherichia coli
Pulse-chase protein production
Yeast
High Efficiency Transformation
Simple Yeast Lysate (for Western etc.)
T7
Simple lysate protocol from plaque or lysate
Cesium Chloride Purification of T7
Mesoplasma florum
Culture Media
ATCC 1161 Medium
Mix a base broth in a 1 liter 45mm cap bottle the following chemicals:
| Heart Infusion Broth (Difco 0038) | 17.5 g |
| Sucrose | 40 g |
| Agar Noble (Difco 0142) if necessary | 12 g |
| DI Water | 710 ml |
Autoclave at 121C for 20 minutes and cool to room temperture (broth) or 55C (Agar) on a 55C water bath.
Centrifuge 2 x 45 ml of Gibco 18180-059 liquid yeast extract at high speed to remove sediment. Pour only 45 ml into centrifuge bottles, eliminating as much as possible of the sediment in the bottom of the Gibco stock.
Thaw Sigma H-1138 heat inactivated horse serum. For agar, bring both horse serum and centrifuged yeast extract to 37C. Horse serum at 55C will sediment protein.
Assemble 150ml bottle top 0.22μ filter on the 1 liter bottle. For agar, it is helpful to put the bottle on a foam insulating pad to delay cooling. Add by vacuum filtration:
| Horse serum (Sigma H1138) | 200 ml |
| Yeast extract | 90 ml |
| Penicillin G 500,000 U/ml | 400 μl |
| 2% phenol red | 2 ml |
Adjust pH to 7.4 with 100 mM HCl. Measured pH of as-made broth is 7.46, probably close enough.
Phenol red 2% solution must be neutralized with sufficient NaOH to convert it to the sodium salt. The phenol red amount can be adjusted to control the depth of color to taste.
SP-4 (ATCC 988) Medium
Mix a base broth containing:
| Mycoplasma broth base (BBL 111458) | 3.5 g |
| Tryptone | 10 g |
| Peptone | 5.3 g |
| Glucose | 5.0 g |
| DI water | 615 ml |
Adjust pH to 7.5 and autoclave at 121C for 20 minutes.
Centrifuge 2 x 17.5 ml of liquid yeast extract (Gibco 18180-059) at high speed to remove sediment.
Mix 2 g of Yeastolate (Difco 5577) in 100 ml DI water.
Sterile filter into the cooled medium:
| penicillin G 500,000 U/ml | 400 μl |
| 2% phenol red | 2 ml |
| 2% Yeastolate (Difco 5577) | 100 ml |
| Newborn calf serum | 170 ml |
| liquid yeast extract | 35 ml |
| 10x CMRL-1066 solution | 50 ml |
In vitro
Nucleic acids
Quantification of nucleic acids
Denaturing acrylamide gel purification of nucleic acids
DNA
Phosphatase treatment of linearized vector
'Round-the-horn site-directed mutagenesis
RNA
Northern Blot, 32P End-Labeled Probes
Probe Prep, 32P End-Labeled Probes
In vitro transcription with T7 RNA polymerase
Protein
Microfluidics
Order a Microfluidic Chip from the Foundry
Operate Microfluidic Chemostat
Microscopy
Flow Cytometry
Miscellaneous
Counteracting evaporation from wells in a Victor3 plate reader
