electroporation (to make cDNA libraries)
Rex L. Chisholm
chisholm at .cmsbio.nwu.edu
Tue Dec 15 09:00:48 EST 1992
djt2 at po.CWRU.Edu (Dennis J. Templeton) writes:
>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
>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
>Electroporation of this (in several batches) has the potential to generate
>10e8 to 10e9 clones.
>From a practical standpoint, the high electroporation efficiencies are at
>very low levels of supercoiled plasmid. We have never gotten more than
>10e6 clones from a single pulse, but 10 pulses of a ligation are practical,
>and 10e6 is plenty of cDNA's anyway.
>Input from any who has made libraries this way would be appreciated, and
>those contemplating it should look at the article in this month's
>BioTechniques (13, Nr6. 862-864, 1992) wherein the authors describe using
>pBluescript to make T-tailed vector to clone cDNA's a al Okayama and Berg.
We have been trying to do the same thing recently but have been unsuccessful
at achieving any significant level of introduction of lambda DNA, even though
plasmid gives us >10e9 transformants/microgram. Anyone else have luck with
lambda DNA and electroporation?
chisholm at worms.cmsbio.nwu.edu
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