Paulsson:Protocols: Difference between revisions
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doubling time for E. coli reported to be 45 (-/+10) min at 32 degrees<br> | doubling time for E. coli reported to be 45 (-/+10) min at 32 degrees<br> | ||
for microscope pad, dissolve low melt agarose in MSC (1.5%)<br> | for microscope pad, dissolve low melt agarose in MSC (1.5%)<br> | ||
*AB media - free of precipitated salts (from Clark & Maaloe, J.Mol.Bio. v23:99-112, 1967.) | |||
Solution A: 2.0g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>, 6.0g Na<sub>2</sub>HPO<sub>4</sub>, 3.0g NaCl, 0.011g Na<sub>2</sub>SO<sub>4</sub>, dissolved in 200ml of water.<br> | |||
Solution B: 0.2g MgCl<sub>2</sub>, 0.01g CaCl<sub>2</sub>, 0.0005g FeCl<sub>3</sub>:7H<sub>2</sub>O in 800ml of water. | |||
I usually dissolve a measurable amount of FeCl<sub>3</sub>:7H<sub>2</sub>O and keep this as stock solution. | |||
===Plasmid Biology=== | ===Plasmid Biology=== | ||
Revision as of 12:28, 15 October 2009
Many excellent protocols can be found in Current Protocols in Molecular Biology, available through the Countway Digital Library.
Please contribute to this page!!!
Preparation of electrocompetent E.coli
Preparation of electrocompetent E.coli, v2
Electroporation of E.coli
Measuring DNA replication with 3H-thymidine
Shipping Information
Nanodrop
Plate Reader
Getting Started In the Lab
Pipetting 101
general
what volume, which tool
accuracy and calibration (cleaning)
Sterile technique
Nurturing Microbes
preservation growth conditions genotypes antibiotics
Media Preparation
Current Protocols in Molecular Biology article on mimimal, rich, solid and agar media for E. coli
Media:Ecoli_preparation_media.pdf
- MSC media (from Rosenfeld et al., Science v307, 25 March 2005, SOM)
M9 salts
0.6% succinate
0.01% casamino acids
0.15 ug/ml biotin
1.5 uM thiamine
doubling time for E. coli reported to be 45 (-/+10) min at 32 degrees
for microscope pad, dissolve low melt agarose in MSC (1.5%)
- AB media - free of precipitated salts (from Clark & Maaloe, J.Mol.Bio. v23:99-112, 1967.)
Solution A: 2.0g (NH4)2SO4, 6.0g Na2HPO4, 3.0g NaCl, 0.011g Na2SO4, dissolved in 200ml of water.
Solution B: 0.2g MgCl2, 0.01g CaCl2, 0.0005g FeCl3:7H2O in 800ml of water.
I usually dissolve a measurable amount of FeCl3:7H2O and keep this as stock solution.
Plasmid Biology
definitions transformation isolation segragation incompatability copy number selection
Manipulating DNA
Cloning Synthetic Oligonucleotide Cassettes
Quick Change mutagenesis
Colony PCR
Agarose Gels
- For small casting tray, 0.6g agarose + 60 ml TAE, microwave 50-60 sec, add 3ul EthBr stock, let stand for 7 min before pouring
Narrow wells (15 lanes): 1.5 x 2.59mm, ~19ul volume
Wide wells (8 lanes): 1.5 x 5.54mm, ~40ul volume
- For large casting tray, 150ml volume, microwave 80-90 sec, add 7.5ul EthBr stock, let stand 10min before pouring
Narrow wells (20 lanes): 1.5 x ##mm, ~##ul volume
Wide wells ( lanes): 1.5 x ##mm, ~##ul volume
Multi-channel pipet compatible wells (26 lanes): 1.5 x mm, ~##ul volume
