Biology Reference
In-Depth Information
H
2
O to limit the ions present in the sample that could result in arcing during
electroporation.
a. Determine the volume and concentration of the DNA sample.
b. Add 1/10 volume of 3
M
sodium acetate (pH 5.0).
c. Add 2.5 volumes of cold 100% ethanol.
d. Mix and incubate at 4°C for1horovernight at -80°C.
e. Pellet the precipitated DNA by centrifugation (4°C, 16,000 ×
g
, 15 min).
f. Remove supernatant.
g Wash the pellet one to three times with 1 mL cold 70% ethanol.
h Aspirate supernatant and dry sample in speed vacuum or laminar flow hood.
i. Thoroughly resuspend the pellet in dH
2
O with a pipettor, such that the final
concentration is approximately 5 μg/μL.
j. Store concentrated plasmid DNA at 4°C for a few hours or at -20°C for
several days.
13. The efficiency of electroporation can be estimated by monitoring the time
constant (TC) associated with each pulse. First, electrocompetent spirochetes
should be electroporated without additional plasmid DNA. The TC generated
from the pulse of spirochetes alone should be recorded and should be between
4.0 and 4.7 ms when using 2.5 kV, 25 μF, and 200 . If the spirochetes alone
generate an arc, or the TC is 2-3 ms, the cells must be washed again. Addition of
plasmid should not alter the TC significantly from the spirochete-alone control.
If addition of plasmid DNA results in arcing, either the plasmid was not de-
salted properly or it is too concentrated. First try using less plasmid. If arcing
still occurs, the plasmid must be re-precipitated and washed more thoroughly.
pBhSV-2 is a very useful positive control plasmid for monitoring the efficiency
of transformation. The standard method for quantifying transformation efficiency
(number of transformants/μg DNA) is not applicable with liquid culture and
cloning by limiting dilution. One modification that increases the efficiency of
transformation is to linearize the inactivation plasmid DNA prior to precipitation
and electro-transformation.
14. Counting the spirochetes with a dark-field microscope helps to approximate the
concentration of spirochetes and the number of 10-fold serial dilutions required.
Following limiting dilution, incubate at 34°C and monitor the red-to-yellow color
change provided by the phenol red pH indicator in mBSK-c. The time required
for color change is shorter in the more concentrated wells and is apparent in
approximately 3-5 days. Do not use color change to conclude the presence or
absence of growth in a particular well. Analyze individual wells by dark-field
microscopy and Poisson distribution statistics to determine clonality.
Acknowledgments
We thank Merry Schrumpf, Kevin Lawrence, Aimee Giessler, and Gail Sylva
for excellent technical assistance; Patti Rosa, Jim Bono, Abe Elias, and Philip
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