TRANSFER OF PLASMIDS INTO BACTERIA

David J. Westenberg djwesten at coos.dartmouth.edu
Fri Mar 3 12:29:58 EST 1995


leach at bu.edu (Martin D. Leach) writes:

>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.

Even less will work if necessary.

>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)

The above manuals have simple almost fool-proof methods for making your own 
competent cells and how to use them that will work just fine without 
having to buy competent cells.  You just need to get your hands on a 
good transforming strain such as DH5alpha (but there are a lot of 
strains which are almost as good).

(standard transformation protocol deleted)

>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)

This could result in wasting a lot of your precious plasmid sample.  

*Not all plasmids code for ampicillin resistance*  Check with the 
source of your plasmid to find out what plasmid vector was used, which  
antibiotic resistance it codes for and at what concentration to use for 
selection.

>Grow overnight in 37 C incubator (plates inverted)




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