TITLE: Chlamydomonas reinhardtii nuclear transformation by electroporation.
AUTHORS: João Vitor Molino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocols describe the steps required for nuclear transformation of </span><span style = "font-style:italic;">Chlamydomonas reinhardtii </span><span>by electroporation.</span></div><br/><div class = "text-block"><a href="https://youtu.be/VP1tSdNXvGY" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Here</span></a><span> you can find a video following the protocol. </span></div></div>

[STEPS]
?. [DNA Preparation]
Digest a large enough amount of plasmid. The goal is to have a concentrated digested sample in the range of 250-700 ng/uL.Select the appropiate enzymes for linearization. Usually, restrictions sites in flanking position to the expression cassete.Mix all components for digestion   . Digest for   at   .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. Quantitate by absorbance measurement (i.e. Nanodrop).Mix all components for digestion  . Digest for  at  .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. Quantitate by absorbance measurement (i.e. Nanodrop).  AB1ComponentAmount210X Cutsmart NEB6.0 uL3XbaI | NEB 20 U/uL3.0 uL4KpnI HF | NEB 20 U/uL3.0 uL5Plasmid 1219.9 ng/uL40 uL6ddH20, Molecular grade8.0 uL
[uncut vector]
37 °C
AB1ComponentAmount210X Cutsmart NEB6.0 uL3XbaI | NEB 20 U/uL3.0 uL4KpnI HF | NEB 20 U/uL3.0 uL5Plasmid 1219.9 ng/uL40 uL6ddH20, Molecular grade8.0 uL
Result example
?. [Cells preparation]
Aseptically inoculate  with wild type cells. Either by scrappeing cells of a plate with a inocculating loop or from a previous cultured cells.Incubate at  , under constant shaking (~150-180 RPM) and light (60-80 µmols de photons/m2s) until a cell density from  to  is reached.Pellet cells in centrifuge tubes. Separate culture in sufficient amount of sterile 50mL centrifuge tubes or larger volume tubes, and centrifuge for .
[Tap media]
25 °C
3
6
Centrifuge: 2000 34, 10 min, 25 10
Culture at 3x106 cells/mL usually yield 12-13 transformations.
?. [Transformation]
Add cutted vector to the bottom of the electroporation cuvette. Typically from   to Add   (at approximatelly ) 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).                                                                                                      Incubate cells with DNA  for Wipe cuvette (to remove condensated water) and electroporate (Table Electroporation).Let it recover for   on the cuvetteAdd cells to   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   on rocker or shaker at 50 rpm   ambient light.     Pellet cells by centrifuging for   Aseptically poor off supernatant. Add   to pelet. Gently re-suspend cells and pipette onto 2 plates with appropriate antibiotics. ie.   per plate, and let it dry a,septically without plate cover.Spread cells evenly over the plate with a innoculation loop. Avoid spreading to the borders. Let it recover for  on the cuvetteAdd cells to  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  on rocker or shaker at 50 rpm  ambient light.  Pellet cells by centrifuging for Aseptically poor off supernatant. Add  to pelet. Gently re-suspend cells and pipette onto 2 plates with appropriate antibiotics. ie.  per plate, and let it dry a,septically without plate cover.Use parafilm to block evaporation and place plates under constant light (60 µmols de photons/m2s), . Colonies should be visible in 5-7 days. Table Electroporation - Settings  AB1Voltage800 V2Time Constant20 ms3Cuvette gap4 mmTypical output after electroporation  ABCD1Time constant (ms)Voltage (V)Capacitance (uF)Resistance ( Ohms)220.178850650320.478950600419.878950550
[cutted vector]
[cutted vector]
[ressuspended cells ]
on ice
[TAP/40mM sucrose, pH 7.0]
0 Room temperature
Centrifuge: 2000 34, 10 min, 25 10
[TAP/40mM sucrose]
[cells ressuspended]
25 °C
AB1Voltage800 V2Time Constant20 ms3Cuvette gap4 mm
ABCD1Time constant (ms)Voltage (V)Capacitance (uF)Resistance ( Ohms)220.178850650320.478950600419.878950550
Green colonies should appear in the plate as in the pictures below.