electroporation (to make cDNA libraries)

Dennis J. Templeton djt2 at po.CWRU.Edu
Mon Dec 14 17:01:04 EST 1992


In a previous article, pakapke at IASTATE.EDU () says:

>
>     We would like to get away from in vitro packaging our phage DNA and
>simply electroporate it into the cell directly.  Anyone in net-land had
>experience doing this?  Are there any references published using this
>technique to produce a phage library?
>

Hmm.  I don't have a direct answer for you, but I would be cautious about
trying to electroporate DNA as large as the concatemers of lambda you get
after ligation.

On the other hand, we spent months making a good cDNA library out of
LambdaZAP, THEN got experienced in electroporation of coli.  Given the high
electrocompetency rate (10e10 is not unrealistic) it seems we might have
been just as successful at making our cDNA library in pBluescript.

Here are some numbers:

5 ug A+ RNA, makes about 5 ug DS cDNA
Ligating 1 ug of this into 0.5 ug of *prepared* (see below) pBluescript
	should result in about 0.1 ug of circularized vector (probably
	more)
Electroporation of this (in several batches) has the potential to generate
10e8 to 10e9 clones.  



More information about the Methods mailing list