"Thermal" means "heat", but the important bit for this one is step 4,
electroporation. If you've got access to a Bio-Rad GenePulser then
that's awesome; there've been some discussions of DIY electroporation
using piezo lighters on this mailing list, but I don't know if anyone
has successfully transformed bacteria with one yet. (Simon? Nathan? I
seem to recall y'all discussing this. Also I've owed John Griessen an
email about this for months now, but real life has intervened more
than it typically does.)
Lactobacilli aren't terribly amenable to heat shock. There are some
protocols in the patent literature involving PEG but they're
apparently quite touchy.
Cheers,
--mlp
On Tue, Mar 6, 2012 at 8:01 PM, Petfixer71 <afrishman71@hotmail.com> wrote:
> |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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