[DIYbio] Most first solo project ...The Yogurt Hack....advice./Feedback..

|One of the first "projects" I want to try on my own (after doing some
basic "Kit" transformations) is "hacking yogurt".

I have a couple question regarding the below protocol:

1) Thermal Shock- is suggested for this transformation.....Can I do a
"heat shock" instead? with same results?

2) Plasmids- pJK650 and pLEM415....can I purchase these plasmids
online? I need a resource for ordering plasmids?

3) Can the plasmids be custom ordered? with DNA of my choice?

I would love some feedback.

Thanks,

Andrew

Below is the protocol:
_______________________________________________________________

Materials

List reagents, supplies and equipment necessary to perform the
protocol here. For those materials which have their own OWW pages,
link to that page. Alternatively, links to the suppliers' page on that
material are also appropriate.

* MRS media
* Electroporation Buffer:(0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4;
PH 6)
* Milk Medium: (0.2 M sucrose, 5% skim milk, 0.1% yeast extract,
1% Casamino Acids, 25mM MgCl2)
* Erythromycin, Chloramphenicol, other antibiotics may be needed
for selection
* Gene Pulser and Pulse Controller Electrophoration apparatus
* Gaspak or method to generate anaerobic conditions
* Strain of compatible Lactobacillus - For list of compatible
strains/plasmids, please click [here]

Procedure

Estimated time for procedure: 3-4 days

1. CELL CULTURE
1. Inoculate serial dilutions of fresh bacterial culture into
100 ml of MRS containing 0.1% glycine and incubate at 42°C overnight.
2. Harvest 10 ml of culture cells at beginning of stationary
phase (optical density at 600 nm, 1.7) by centrifugation (need ~2.3mL
of culture per electroporation)
2. WASH BUFFER
1. `Wash bacteria once with 100 ml of cold electroporation
buffer (EB) (0.4 M sucrose, 1 mM MgCl2, 5 mM Kh2PO4; PH 6)
2. Wash bacteria twice with 30 ml of cold EB
3. THERMAL SHOCK
1. Resuspend cells in EB to an optical density at 600 nm of
about 50
2. Incubate cell suspension at 45°C for 20 min then keep on
ice for 10 min
4. ELECTRICAL PULSE
1. Mix 80 ?l of cell suspension with 0.3 to 2 ?g of plasmid
DNA
2. Subject sample to a 1-kV, 800-Ω, 25-?F electric pulse in a
0.2-cm cuvette by using a Gene Pulser and a Pulse Controller
apparatus.
5. EXPRESSION
1. Immediately add 2 milliliters of milk medium (0.2 M
sucrose, 5% skim milk, 0.1% yeast extract, 1% Casamino Acids, 25mM
MgCl2)

*Derek Ju 04:28, 29 October 2008 (EDT): I have found that adding MRS
works just as well (and saves the step of making the milk medium)

1. PLATING, SELECTION
1. Incubate cells for 3h at 37°C before plating on MRS agar
supplemented with antibiotics. Add antibiotic erythromycin at
concentration 7.5 ?g/ml and chloramphenicol at concentration 7.5 ?g/ml
depending on plasmid resistance gene
2. Incubate plates at 37°C for 2 to 3 days under anaerobic
conditions in jars containing GasPak

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