Electroporation protocol

Dennis Wykoff dwykoff at andrew.stanford.edu
Thu Jun 11 16:29:06 EST 1998


Dear Chlamydomonas Community,

 Don Weeks, who has been visiting the Grossman laboratory, and I have
 sat down and put together our protocol on how to transform Chlamy
 using the electroporation technique of Kosuke Shimogawara.  Our
 protocol differs in some of the details, but essentially follows his
 protocol.  We have optimized the protocol for the Biorad Gene Pulsar
 I.  I hope it helps get you over the hurdle of trying
 electroporation.

Protocol:

1. Grow cells to approximately 1-5 x 10^6 cells/mL
 Cells must be in mid log.  If your strain doesn't grow well, you may
 want to back cross it before attempting transformation.  Furthermore,
 it is vital that the strain is wallless (either by mutation or
 autolysin treatment). Cwd and cw15 seem to be good for this.


2. Add 1:2000 dilution of sterile 10% Tween-20 (This is only needed
when you do not have a lot of cells).  The tween helps to pellet cells
(e.g. for 100 mL of cells I add 50 ul.)

3. Spin cells down as gently as possible between 2000 and 5000g or 5
min.  Remember to keep everything sterile!

4. Remove all supernatant and resuspend in TAP+60 mM sucrose.
 If you are using a different medium than TAP, you may want to change
 the concentration of sucrose. We have found that the sucrose
 concentration is flexible (anywhere between 40-60 mM).  It is
 important that the cells are in a medium relatively isotonic to the
 cytoplasm, so if you are using a medium other than TAP, you may want
 to adjust the sucrose concentration accordingly.  Don has suggested
 that you may want to use lactose, if contamination is a problem, but
 this hasn't been tested. Generally, we resuspend the cells in 1/100th
 volume, but for the highest absolute number of transformants, it
 helps to resuspend in the least amount of medium required (i.e. 5 X
 10^8 cells/mL).

5. Place the resuspended cells on ice and prepare electroporation
cuvettes.  We use cuvettes that have a 0.4 cm gap.  We (and Kosuke)
have used 0.1 cm cuvettes and they appear to work just as well,
although the voltage must be adjusted (see below). Make sure the
cuvettes don't leak!  If you reuse cuvettes (which we do) when you are
finished wash the cuvettes thoroughly (rinsing with a jet of water
from a P200 pipet tip connected to a water hose works well) and just
before use sterilize in ethanol for 30 minutes and then allow them to
dry in a sterile hood.  Store the cuvettes under sterile conditions
until use.

To prepare the cuvettes, we usually pipet in 5 ul of 10 mg/mL single
stranded, sonicated, heat-denatured salmon sperm DNA into a cuvette
and then pipet in the plasmid DNA so that there is 2.5 ug DNA per
cuvette. The amount of DNA is flexible.  It appears that
transformation efficiency is pretty linear between .2 and 2.5 ug of
DNA, and it maybe linear at even lower concentrations.

6. Add 250 ul of the cells and place the cuvette into a chamber
that will cool the cuvette to 15oC.  Let this cool for at least 2
minutes (however it can cool for 30 minutes and not be affected).  We
use a water bath, but probably any set up will work that will cool the
cuvettes for a few of minutes. Then electroporate.

7. Electroporation
All electroporators are different.  Our conditions use a Biorad Gene
Pulsar I electroporator, with the capacitance set at 3 uF and the
voltage at 1.8 kV (giving a V/cm of 4500).  Many setups will give
similar conditions.  We have found that you do not need a shunt
resistor with our device.  In fact, a 300kOhm resistor lowered the
efficiency 4-5 fold.  Expect a time constant for these conditions of
1.2-1.4 ms. (Note Kosuke's set up yields very different time
constants, around 5ms with 25 uF, 4.5 kV/cm; under these conditions we
only get a few transformants per trial).  Return the cuvette to the
15oC chamber. Plate the cells within an hour of electroporation.


8. Plating
Using previously prepared cornstarch, pipet 1-1.5 mL of cornstarch
onto a plate and then pipet an aliquot of the electroporation mixture
into the "puddle" of starch (with cwd arg7 strain and 2.5 ug DNA,
plating 5ul of transformation mix usually gives 200-400 colonies). If
necessary, add more TAP+sucrose to get the cornstarch evenly spread on
the plate.  To spread the cells and cornstarch tilt the plate slightly
and rock the plate gently (practice makes perfect).  It helps if the
plates are relatively fresh (1 week).  Let the plates dry in a sterile
hood (until there is no obvious liquid on the plate and you can tilt
the plate without the cornstarch moving...approx 30 min), and then
place the plates in low light (5 uE). Twenty-four hours later move to
growth conditions (80 uE).

Cornstarch mixture: Using a box of Kingsford (or any brand)
cornstarch, measure out 12 g of cornstarch into a 50 ml capped tube.
Add 95% ethanol to this until the volume is 50 ml.  Mix well, and make
sure that the cornstarch is well suspended in this mixture.  Allow the
cornstarch to settle.  Mix and settle 3 times over the next 24 hours.
The cornstarch can be stored for long periods in ethanol (many
months).  The same day as electroporation, pour off the ethanol and
wash the cornstarch with TAP+sucrose at least three times. We mix the
cornstarch with the TAP+sucrose (and resuspend completely) and then
spin down the cornstarch in a table top centrifuge, and repeat.  Bring
the final volume of the cornstarch suspension to 45-50 ml.  One ml of
this 25% cornstarch solution is more than enough for each plate.

We consistently obtain about 1500-2000 transformants per trial with
the nit1 gene (pMN24), and 10000 transformants per trial with
linearized ARG7 (pJD67).  This can be further optimized as Kosuke
routinely obtains 20-30,000 transformants per ug of DNA.  Note when
this procedure was performed using 25 uF instead of 3 uF, we obtained
about 100 transformants per trial.  One good way to judge if you have
used too strong of an electric current, is that cells will sediment
and "clump" together when they have been over electroporated. Don has
found that allowing for a recovery period over night leads to a
significant increase in the number of transformants, although some of
this increase is due to cell division.

In conclusion following this simplified protocol should yield a
significant number of transformants.  If you wish to optimize, we
suggest:
 1. Backcrossing the strain to get it into a very healthy cw
background.
 2. Allowing for a recovery after electroporation.
 3. Varying the voltage,resistance, and capacitance of
electroporation.
 4. Varying the temperature of electroporation or sucrose
concentration.


Feel free to email me with any questions,

Dennis Wykoff

dwykoff at andrew.stanford.edu

Grossman Laboratory






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