TRANSFER OF PLASMIDS INTO BACTERIA

Martin D. Leach leach at bu.edu
Fri Mar 3 10:57:19 EST 1995


In article <9503021338.aa06191 at etsuodt.etsu.edu>, betts at ORION.ETSU.EDU
(GORDON BETTS) wrote:

> I have obtained a pair of plasmids (a whole 1 ul of each) and now must do
> something with them.  Since I am new to this area of work, I'm hesitant to
> just jump in.  So, does anyone have good advice on how to do a one-time,
> not-a-chance-of-failure transfection into bacteria?  What's a good vector
> for this?
> Thanks,

1. It is a transformation and not a transfection.

2. What is the concentration of the DNA...? For a single transformation
only 5-100 ng of DNA is required...so you can dilute your sample
accordingly and perform a number and not just a hit or miss experiment.

3. Try reading Maniatis: Lab Cloning Manual or try Current Protocols..

Very briefly:

Obtain some sub cloning efficiency DH5alpha cells (e.g. Gibco BRL)
thaw one tube of cells on ice for 20mins (600 ul = 6 assays)
mix cells gently by pippeting and place 100 ul of cells in precooled
polypropylene tube (see what is recommended by the protocol obtained with
the cells)

Add 5-100ng of DNA per 100ul aliquot of cells (you cannot exceed 5%
volume) so no more than 5ul of DNA can be added to 100ul of cells. Mix DNA
with cells and place on ice for 30'.

then transfer to 42C for 45sec then replace on ice for 2mins

(note...this timing is dependant on the tubes you are using)

Add 900ul of SOC medium (i use Luria Broth instead) to the cells and
incubate the mixture for 60' in a shaking 37C incubator/waterbath
(250rpm).

You can then streak the cells onto Luria Agar plates containing 25ug/ml
ampillicin. or add a number of aliquots to a number of plates (e.g.
25,50,100,200,400 ul to 5 plates....and allow them to dry onto the plates
by incubating them right-side up with lids off the dish for a short
period. (this is the simplest way for a novice)

Grow overnight in 37 C incubator (plates inverted)

...for more assisstance contact GibCo BRL and ask them about
transformations using their DH5alpha competent cells....they should be
able to FAX/point you in the right direction...

---no affiliation with any of above....I may be related to Uncle Luria
Agar though....;)

Martin

-- 
.....          Martin Leach                Email:leach at bu.edu 
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