Paulsson:Protocols

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List of protocols we should have

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A few 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 miimal, 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 salts - free of precipitated salts (from Clark & Maaloe, J.Mol.Bio. v23:99-112, 1967.)

Solution A: 2.0 g (NH₄)₂SO₄, 6.0 g Na₂HPO₄, 3.0 g NaCl, 0.011g Na₂SO₄, dissolved in 200 ml of water.
Solution B: 0.2 g MgCl₂, 0.01 g CaCl₂, 0.0005 g FeCl₃:7H₂O in 800 ml of water.
I typically dissolve a measurable amount of FeCl₃:7H₂O and keep this as stock solution (PerM).

A and B are mixed after autoclaving.

• Casamino acids

Vary by manufacturer!!! Fluka "Amicase" is a casein hydrosylate, but different than BD Bacto "Casamino Acids". A 10% solution of Fluka amicase (cat.# 82514) will go into solution with autoclaving, but will then precipitates upon cooling. BD casamino acids (BD cat# 223050) goes into solution quite easily at 10% w/v. (Fluka CAS also darker/yellower than BD.) Labscientific "Casmamino acids" at 10% almost stays fully in solution and is almost as dark/yellow as Fluka amicase.

Make 10% w/v solution. Autoclave. Add to defined salt solutions (eg. M9, AB) to 0.1% final.

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

• 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 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

Polyacrylamide Gels

Chemistry of polyacrylamide gels