Chlamydomonas reinhardtii nuclear transformation by electroporation.

Digest a large enough amount of plasmid. The goal is to have a concentrated digested sample in the range of 250-700 ng/uL.

1. Select the appropiate enzymes for linearization. Usually, restrictions sites in flanking position to the expression cassete.
2. Mix all components for digestion 40µg . Digest for 6h 0m 0s at 37°C .
3. Column purify digestion (Avoid gel purify, since vector backbone may helps to prevent intracelular DNAses action). * Use a PCR purification kit to purify the digestion reaction.
4. Quantitate by absorbance measurement (i.e. Nanodrop).

A	B
Component	Amount
10X Cutsmart NEB	6.0 uL
XbaI	NEB 20 U/uL
KpnI HF	NEB 20 U/uL
Plasmid 1219.9 ng/uL	40 uL
ddH20, Molecular grade	8.0 uL

Typical reaction setup

Aseptically inoculate 250mL with wild type cells. Either by scrappeing cells of a plate with a inocculating loop or from a previous cultured cells.1. Incubate at 25°C , under constant shaking (~150-180 RPM) and light (60-80 µmols de photons/m²s) until a cell density from 3x 10^6 cells/mL to 6x 10^6 cells/mL is reached. Pellet cells in centrifuge tubes. Separate culture in sufficient amount of sterile 50mL centrifuge tubes or larger volume tubes, and centrifuge for 2000x g,25°C.  

1. Genttly ressupend cells at 3-6-108 cells/mL ⁸ cells/mL in Transformation Buffer.

Add cutted vector to the bottom of the electroporation cuvette. Typically from 250ng to 1000ng Add 250µL (at approximatelly 3x 10^8 cells/mL) to each cuvette. Pippet up and down on DNA sample. Flick cuvette to mix DNA and cells. Shake cells to the bottom of the cuvette. Also add no DNA control (Elution buffer or water).

1. Incubate cells with DNA On ice for 0h 10m 0s
2. Wipe cuvette (to remove condensated water) and electroporate (Table Electroporation).
3. Let it recover for 0h 10m 0s on the cuvette
4. Add cells to 10mL inside sterile 50mL centrifuge tubes. Gently transfer cells from cuvette to TAP/40 mM sucrose. Rinse cubette with TAP/40 mM sucrose to transfer any remaining cells. Incubate at Room temperature on rocker or shaker at 50 rpm ambient light.

1. Pellet cells by centrifuging for 2000x g,25°C
2. Aseptically poor off supernatant. Add 300µL to pelet. Gently re-suspend cells and pipette onto 2 plates with appropriate antibiotics. ie. 200µL per plate, and let it dry a,septically without plate cover.
3. Spread cells evenly over the plate with a innoculation loop. Avoid spreading to the borders.
4. Use parafilm to block evaporation and place plates under constant light (60 µmols de photons/m²s), 25°C. Colonies should be visible in 5-7 days.

Table Electroporation - Settings

A	B
Voltage	800 V
Time Constant	20 ms
Cuvette gap	4 mm

Equipment

Value	Label
Gene Pulser Xcell Electroporation Systems	NAME
Electroporator	TYPE
Biorad	BRAND
1652660	SKU

Typical output after electroporation

A	B	C	D
Time constant (ms)	Voltage (V)	Capacitance (uF)	Resistance ( Ohms)
20.1	788	50	650
20.4	789	50	600
19.8	789	50	550