Bio-Rad MicroPulser™ Electroporator - Manual (Page 28)

Biological
The general symptom addressed in this section is transformation efficiencies that are too
low to detect or too low to be useful. The following is a list of the areas of possible problems
and some suggested solutions.
Problem Possible cause and solution
1. The pulse. Is the pulse actually applied to the sample? At high
voltage with a small-volume (40 µl) sample this is
easy to check. The sample will "twitch" when
pulsed. If you don’t see a twitch, refer to the electrical
troubleshooting section for information on electrical
problems. Also make sure that the cuvette is making
contact with the electrodes at the back of the sample
chamber. If electrodes are broken or corroded call
Bio-Rad for replacements.
Are the amplitude and length of the pulse suffi-
cient?
E. coli requires pulses of approximately 5
msec with field strengths of 12 to 18 kV/cm. S.
cerevisiae requires pulses of approximately 5 msec
with a field strength of ~7.5 kV/cm. There is usually
some cell death with electrical conditions producing
transformation. Survival rates of 20 to 80% are typical.
If no cell death occurs, the pulse is probably too
weak. Conversely if too many cells are killed
(>80%), the pulse is probably too intense and
transformation will probably be poor. To find the
optimum pulse characteristics, use a pulse length
of ~5 msec and test for transformation over a range
of field strengths.
2. The DNA. Check the quantity and quality of the DNA on a gel.
Often, mini-preps contain less DNA than expected.
DNA stored improperly for long periods may be
degraded and lack transforming activity.
Some preparations of DNA may contain substances
that inhibit transformation or are toxic to the cells.
Try to use DNA free of SDS, phenol, etc.
Is the selection appropriate for the marker (and its
level of expression)?
3. The cells. Were the cells harvested at the correct stage in the
growth phase? Bacterial cells generally show the
highest transformation efficiencies when
harvested in the early to mid-log growth phase.
Yeast cells generally show the highest transformation
effiencies when harvested in late log phase.
Different growth conditions may improve
transformation.
Are too many cells killed? The pulse is too intense,
toxic substances are present in DNA or cell
preparations, wrong temperature of electroporation
are all possibilities.
Are the cells transferred to outgrowth medium
immediately after the pulse? For
E. coli this is very
important.
Is the correct selection applied after the recovery
period?
4. The temperature. Are the cuvettes cold?
Is the cuvette holder (slide) prechilled?
If frozen, have the cells been stored properly
(usually in 10-15% glycerol at -70 °C)?
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