Paulsson:Protocols

Many excellent protocols can be found in Current Protocols in Molecular Biology

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

Ordering Oligos
Create a Harvard Biopolymers Facility account: 1. Go to the Biopolymers Facility website 2. On the far left side of the screen click on “New Accounts” 3. Select “Online Account Registration” on the upper middle of the page 4. Select “Create User account” 5. Enter our lab address (WAB 461) 6. Select Johan Paulsson from the PI drop down 7. Submit your account for approval. It should be approved within 24 hours, usually much faster

When you have a BPF account or if you already have one: 1. Again, navigate to the Biopolymers Facility 2. Login on the upper right side, then select “Oligo… Synthesis” on the far left middle of the page 3. Select “Place an Order” 4. Please note the information under “IDT ORDERS” which states >> “You now have access to the IDT BPF Oligo Portal System. Your username is: bpf_(your username), and your password is the same as your normal BPF password.” 5. Click on “IDT Oligo Portal System” 6. Login using the login name and password instructions from Step 4 above. DO NOT USE YOUR OLD IDT LOGIN INFO. 7. Once you are logged in it will be the same IDT website that you are familiar with.

Nurturing Microbes
preservation growth conditions genotypes antibiotics


 * P1 transduction
 * Reusing electroporation cuvettes

Media Preparation
Current Protocols in Molecular Biology article on mimimal, rich, solid and agar media for E. coli [[Media:Ecoli_preparation_media.pdf]]

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%)
 * MSC media (from Rosenfeld et al., Science v307, 25 March 2005, SOM)

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. A and B are mixed after autoclaving.
 * AB media - free of precipitated salts (from Clark & Maaloe, J.Mol.Bio. v23:99-112, 1967.)

I typically dissolve a measurable amount of FeCl3:7H2O and keep this as stock solution (PerM).

see also http://openwetware.org/wiki/AB_medium

http://openwetware.org/wiki/Casamino_acids

Plasmid Biology
definitions transformation isolation segragation incompatability copy number selection

Manipulating DNA
Cloning Synthetic Oligonucleotide Cassettes Quick Change mutagenesis Colony PCR DNA precipitation with glycogen qRT-PCR Isothermal Assembly

Agarose Gels
Narrow wells (15 lanes): 1.5 x 2.59mm, ~19ul volume Wide wells (8 lanes): 1.5 x 5.54mm, ~40ul volume
 * 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 (20 lanes): 1.5 x 4.84mm, ~36ul volume Wide wells (15 lanes): 1.5 x 5.5mm, ~41ul volume Multi-channel pipet compatible wells (26 lanes): 1.5 x 2.9mm, ~21ul volume
 * For large casting tray, 150ml volume, microwave 80-90 sec, add 7.5ul EthBr stock, let stand 10min before pouring

Polyacrylamide Gels
Chemistry of polyacrylamide gels