TITLE: Barcode plasmid library cloning
AUTHORS: Benjamin Emert

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for cloning Time Machine barcode plasmid library. </div></div>

[STEPS]
?. [Gibson assemnbly]
Purify Gibson reaction using PCR clean-up column according to manufacturer's protocol eluting in 20 microliters of
?. Add 40 microliters of DNA binding buffer to 20 microliters of Gibson assembly.
[DNA binding buffer]
[Gibson assembly ]
?. Add 200 microliters DNA wash buffer. Make sure ethanol has been added to wash buffer before use.
[Wash buffer]
?. Centrifuge at 12,000 x g for 1 minute.
?. Repeat wash and centrifugation (steps 2.4 and 2.5)
?. Transfer column to a new eppendorf or collection tube then centrifuge at 12,000 x g for 2 minutes to dry column
Sometime residual liquid can collect on a lip inside the column. It may be helpful to rotate the column 180º after a 1 minute spin, then repeat another 1 minute spin to get rid of this liquid.
?. Add 10 microliter of nuclease free water pre-heated to 50ºC to column and incubate for ≥ 2 minutes
[nuclease free water]
[Pre-heat water]
[Incubate]
?. [Electroporation]
Elecroporation of competent cells
?. [Electroporation]
Thaw Lucigen Endura electrocompetent cells on ice. Each DUO vials contains 50uL of cells, enough for 2 electroporations. Use one reaction's worth of cells to electroporate the negative control Gibson assembly.
?. Centrifuge at 12,000 x g for 1 minute to elute
?. Optionally nanodrop the recovered DNA. Expected concentration is 10-15ng/uL.
?. Re-load eluant and re-spin.
?. [Electroporation]
If using Biorad Gene Pulser, pre-chill cuvettes to 4ºC.
[Pre-chill electroporation cuvettes]
?. [Electroporation]
Pre-warm Lucigen recovery media to 37ºC
[Pre-warm recovery media]
?. [Gibson assembly]
?. [Gibson assemnbly]
Perform Gibson assembly usiing 100ng linearized vector and 5:1 molar excess of ssDNA template per 20 microliter reaction.
?. [Gibson assembly]
?. [Gibson assembly]
?. [Gibson assemnbly]
Combine 100ng linearized vector with ~21.7 fmol ssDNA template (1.08 microliters of 100nM oligo) to a final volume of 10 microliters in nuclease free water. In parallel, perform a negative control reaction with plasmid and nuclease free water without the ssDNA insert. Keep on ice.
[Linearized vector]
[ssDNA oligo (160nt)]
[Final volume in NF water]
?. [Gibson assemnbly]
Thaw Gibson assembly master mix on ice then add 10 microliters to plasmid plus insert. Pipette mix then spin down if needed.
[Gibson Assembly master mix]
?. [Gibson assemnbly]
Pre-heat thermal cycler to 50ºC with lid set to 65ºC.
[Pre-heat thermal cycler]
?. [Gibson assemnbly]
Incubate on thermal cycler at 50ºC for 1 hour.
50 °C
[incubation]
?. [Electroporation]
Pipet vial of electrocompetent cells into 2 25 microliter aliquots.
?. [Electroporation]
Add 2 microliters of cleaned Gibson assembly to 25 microliters of electrocompetent cells.  per
[Gibson assembly]
[electrocompetent cells]
?. [Electroporation]
Transfer electrocompetent cells to pre-chilled cuvette. Keep on ice.
?. [Electroporation]
Using the GenePulserXCell, electroporate with the following manual pulse setting: PL - 25msec, C - 10uF, PC- 600Ohms, V-1800
Pulse length - 25msec, Capacitance - 10 microFaraday,  Resistance - 600 ohms, Voltage - 1800 Volts.
Expected time constant around 5-6 msec.
?. [Electroporation]
Immediately add 975 microliters of pre-warmed recovery media to cuvette then transfer to clean eppendorf or vial.
?. [Electroporation]
Incubate on shaker at 250 rpm at 37ºC for 1 hour.
37 °C
[On shaker at 250rpm]
?. [Electroporation]
Plate dilutions of electroporated cells for plating and calculating electroporation efficiency. Prepare first dilution by adding  to Prepare second dilution by adding  to Plate  on pre-warmed LB/ampicillin agar plate for a 1:1000 dilution plate. Plate  on pre-warmed LB/Ampicllin agar plate for a 1:10,000 dilution plate.
[electroporated cells]
[LB Broth]
[First dilution ]
[LB broth]
[first dilution]
[second dilution]
After incubating the plates overnight, expect to see >1,000 colonies on the 1:1000 dilution plate and >200 colonies on the 1:10,000 plate.
?. [Electroporation]
Add the remainder of the electroporated cells to 150mL LB broth containing 100ug/mL ampicillin. Incubate 12-14 hours on shaker at 32ºC.  -
[LB broth]
32 °C
[shaker]
?. [Electroporation]
Pellet the overnight culture by centrifugation in 50mL falcon tubes then store pellets at -20ºC or proceed immediately with maxiprep.  Pellet e. coli cultures.
4 °C
[Store e. coli pellets]
If centrifuge capable of 6,000 x g is unavailable, can pellet at ≥3,000 x g.
?. Maxiprep barcode plasmid library.
?. PCR screen colonies to confirm barcode insertion.