Variability in Electroporating cDNAs

ALASTAIR MACKAY amackay at WELCHLINK.WELCH.JHU.EDU
Tue Feb 7 23:25:50 EST 1995


Dear Netters,
     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




More information about the Methods mailing list