Variability in Electroporating cDNAs
amackay at WELCHLINK.WELCH.JHU.EDU
Tue Feb 7 23:25:50 EST 1995
I use electroporation to transfect chicken cDNAs into
mammalian cells (HeLas and LL-CPKs). The only vector I have used is
pECE, which uses the SV40 early promoter and also has a 3' LTR.
The problem is variability. Usually, 20% to 40% of cells express
protein 18 hr after electroporation, as judged by immunofluorescence.
Thsi is more than enough for my purposes. When the transfection is
particularly good, 70% - 80% of the cells are expressing protein When
it's bad, about 0.1% of cells express.
I know there are a lot of potential causes for inter- and intra-
experiment variability with this kind of protocol. I have realized that
a fair part of the variability seems to be due to lot-to-lot variation in
the DNA I use. I use a plasmid encoding the full-length protein
(C1F/pECE) as a positive control, and when I was just about out of CsCl
DNA, I made another batch.
Batch 1 C1F/pECE: 50% of cells express.
Batch 2 C1F/pECE: 1% of cells express
As far as I can tell (short of sequencing), both batches are the
same: same host (DH1) transformed with DNA from the same tube, bugs
grown in 500 ml prep, with chloramphenicol amplification (as per
Maniatis), alkaline lysis prep, cesium chloride purification, A260/A280
of about 2.0, both 1 mg/ml in TE, both are mostly supercoiled with some
closed circle, both give the expected patterns by restriction digest.
Both are used to electroporate 2E6 cells in 0.4 ml media in a 4 mm
cuvette, using a Bio-Rad machine at 310 V, 960 uF. Both give tau
readings of about 24 msec.
Looking back on my data, I think that I've made other poor-electro-
porating CsCl preps. With some constructs, I saw nothing at all: due
to unstable protein in the cell and the like, or just to a bad prep of DNA?
Anybody able to shed some light on what I'm doing (variably) wrong?
Please post, or if you e-mail me (amackay at welchlink.welch.jhu.edu) I will
post a summary. Cheers, Alastair Mackay, JHU, Baltimore, Maryland, USA
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