12. Transfer the electroporated cells from the individual cuvettes
into 10 mL of Recovery buffer in 15 mL individual conical
tubes.
13. Incubate the cells for 16–20 h under dim light (2–3 μmol
photons m
À2 s
À1
) to allow for the expression of aph7
00 .
14. Centrifuge the tubes at 600 Â g for 5 min, discard the Recovery buffer by decanting, and suspend the cells using the
remaining medium.
Table 1
Settings for NEPA21 electroporation
Voltage
(V)
Pulse
length
(ms)
Pulse
interval
(ms)
Number
of pulses
Decay
rate (%) Polarity
Poring
pulse
(Pp)
300
8
50
2
40
+
Transfer
pulse
(Tp)
20
50
50
1
À
Æ
300
150
Voltage [V]
20
–20
Decay rate (40%)
Pulse length (8 msec)
Pulse length (50 msec)
Pulse interval (50 msec)
Fig. 1 Schematic of electric pulses delivered by NEPA21. Assignable parameters
in this protocol are represented. Poring pulse (Pp), transfer pulse (Tp), and
polarity-exchanged Tp are shown as black, white, and gray bars, respectively.
Optimal transformation parameters applicable to strain C-9 are indicated in
parentheses
Transformation of Chlamydomonas reinhardtii
159