TITLE: Vibrio natriegens electrocompetent preparation
AUTHORS: David Shis, Weinstock MT, Hesek ED, Wilson CM, Gibson DG

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an approach to electroporate DNA into Vibrio natriegens wt and Vibrio natrigiens sp. Vmax. It's generally pretty similar to canonical electroporation protocols with exception to the wash buffer and temperature of activity.</div><div class = "text-block"><span>Essentially pulled straight from Weinstock </span><span style = "font-style:italic;">et al.</span></div></div>

[STEPS]
?. [Media recipes]
BHI media stock
?. [Media recipes]
BHI + v2 salts- NaCl- KCl- MgCl2- SucroseFilter sterilize or autoclave
204 Milimolar (mM)
4 Milimolar (mM)
23 Milimolar (mM)
680 Milimolar (mM)
?. [Electrocompetent prep]
**** The day before****Inoculate 10 mL [BHI + v2 salts] with V. natriegens overnight culture at 30 °C with agitation at 200 r.p.m.
?. [Electrocompetent prep]
(operator can be at RT, just cells should be chilled)Split outgrowth into two chilled 50-mL falcon tubes and incubated on ice for 15min.
?. [Electrocompetent prep]
(operator can be at RT, just cells should be chilled)Carefully decant supernatant and gently resuspend cell pellett in at lesat 1mL of electroporation buffer (680 mM sucrose, 7 mM K2HPO4, pH 7).
?. [Electrocompetent prep]
**** The day of ****On the following day, prepare 60mL of [BHI + v2 salts] in a baffled flask.Generally, 60mLs of outgrowh yields one electrocompetent cell aliquot.
?. [Electrocompetent prep]
Pellett cells at 4 °C, spinning at 5000 rcf for 10mins.
?. [Electrocompetent prep]
(operator can be at RT, just cells should be chilled)Top off centrifuge tube (in most cases a 50mL falcon tube) with electroporation buffer. Gently mix
?. [Electrocompetent prep]
(on ice)Pellett cells at 4 °C, spinning at 5000 rcf for 10mins.
?. [Electrocompetent prep]
(operator can be at RT, just cells should be chilled)Carefully aspirate away supernatant with a pipette (pellett will be looser than before). Get volume down to around 1-4mL, pellett does not have to be super dry.
?. [Electrocompetent prep]
(operator can be at RT, just cells should be chilled)Repeat steps 8,9,10 at least three times for a minimum of three washes. After pelleting after final wash, carefully pipette off as much of the supernatant as possible.
?. [Electrocompetent prep]
Inoculate media with overnight at a dilution of 1:100 to 1:200 (overnight culture/fresh medium). Incubate culture with shaking at 37 °C shaking at 200r.p.m. until an OD600 of 0.5.
?. [Electrocompetent prep]
(operator can be at RT, just cells should be chilled)Resuspend pellett so final volume of cells + electroporation buffer is about 50-100uL.
?. [Storage]
For long term storage. Snap freeze cell aliquots in dry ice bath or liquid nitrogen. 50uL per electrocompetent aliquot. Store at -80.
?. [Electrocompetent prep]
To check concentration of, generally a 1:1000 inoculation into BHI media should have an OD of 0.15 +/- 0.02. If too dilute, spin down as before and resuspend in a smaller volume.
?. [Electroporation]
Thaw Transfer acqueous electrocompetent aliquot to a chilled 0.1cm gapped cuvette on ice.
?. [Electroporation]
Apply 0.9 kV. So far tested in a biorad micropulsar.Resuspend cells in 500uL of [BHI+v2 salts]
?. [Electroporation]
Outgrow transformation resuspension at(or 30C depending on plasmid) with shaking or rotation.Plate outgrowth onto LB plates with selection.
?. [Media recipes]
Electroporation buffer- sucrose- Potassium phosphate dibasic-     pH7Filter or autoclave to sterilize.
680 Milimolar (mM)
7 Milimolar (mM)