For each LR you are doing, fill out a column in the following table:

For each LR, label a 200 µl strip tube with your initials and tube number.

Into each tube, pipette:

Add 1 µl of TE to each tube

Retrieve an aliquot of LR Clonase from the -80.

Pulse the LR clonase tube in the microfuge to collect the clonase at the bottom.

Add 1 µl of the LR clonase to each LR reaction.

Cap the tubes.

Flick them several times to mix.

Pulse-spin the tubes in the microfuge to collect the liquid at the bottom.

Incubate at room temperature for at least 12 hours and not more than 24 hours.

Retrieve a 5 µl aliquot of proteinase K from the -80 freezer.

Thaw in your fingers, then pulse in the microfuge to collect at the bottom of the tube.

Pipette 1 ul into each of the LR reactions.

Flick several times to mix.

Pulse-spin the tubes in the microcentrifuge.

Incubate at 37° for 15 minutes, or room-temperature for an hour.

Proceed to transformation. Transform 2 µl.

Afterwards, cap the tubes. Write the date on the caps and store in the -20 (in case your transformation failed.)

Make sure the dry bath is set to 42°C and the wells in the block are 1/2 full of DI water

Remove selection plates from the refrigerator. Double-check that they match the selection marker on your plasmid, then place them in the 37° incubator.

Retrieve the DNA to transform.

Fill an ice bucket with ice. Retrieve one tube of competent E. coli per transformation from the -80 and thaw on ice, 3-4 minutes.

While the transformation tubes are thawing, label their tops with something descriptive. Record the labels here:

Add 2 µl DNA from each reaction to a tube of competent cells.

Add 1 µl of the pUC19 transformation control to the positive control tube.

Incubate on ice for 30 minutes.

Heat shock the cells for exactly 30 seconds in the 42° heat block. (Yes, set a timer.)

Place back on ice for 2 minutes.

Add 250 µl SOC to each tube.

Tape the tubes to the platform of a shaker at 37°C and shake at 270 RPM for 60 minutes.

Label the selection plates using the labels you recorded above.

Shake ~10 plating beads onto each plate. (Used innoculation loops instead of beads)

Pipette 100 µl of each transformation onto the corresponding plates.

Cover the plates and shake the beads around to spread the cells out.

Dispose of the beads by tapping them into the waste container.

Incubate the plates upside down overnight in the 37° incubator.

Count the colonies on your positive transformation plate.

Divide the number of colonies by the fraction of the transformation you plated.

Transformation efficiency is expressed in colonies per microgram pUC19. Multiply the number of colonies by the appropriate conversion factor.

Record your transformation efficiency in your (daily) lab notebook.

Warm up the LB to at least room temperature (if it came from the fridge), but not warmer than 37°C

Label one round-bottom culture tube for each miniprep. Use "NAME-1, NAME-2, ..."etc for the naming convention, where NAME is a shortened name of the plasmid (eg, "hEF1a:mKate").

Using a sterile pipette, transfer 5 ml of LB to the mixing container for each culture PLUS 5 ML.

Add antibiotic stock to a final concentration of 1X (1 µl stock for each 1 ml in the mixing container.)

Cap tightly and mix well.

Using a sterile pipette, transfer 5 ml of LB+antibiotic to each round-bottom culture tube.

Squirt ethanol on a pair of foreceps and wipe dry with a Kimwipe.

Use the foreceps to pick up a sterile 200µl pipette tip, scrape a colony off of the plate, and drop the pipette tip in the corresponding tube.

Transfer to an incubating shaker at 37°C and incubate 14-16 hours.

For each culture, label two microcentrifuge tubes on the cap and one blue spin column on the side.

Pipette 1.8 mL of each culture into the corresponding microcentrifuge tubes

Centrifuge at maximum speed (10,000 or 13,000xg) for three minutes.

Aspirate the supernatant, or pour it off into the bleach bucket.

Pipette ANOTHER 1.8 ml of each culture into the corresponding microcentrifuge tubes.

Centrifuge at maximum speed for three minutes.

While the centrifuge is running, move the remaining cultures to 4degC.

Aspirate the supernatant off with the bench aspirator. Be careful not to disturb the pelleted E. coli.

Add 250 µl Buffer P1 to each tube.

Resuspend the E. coli pellet. The preferred way is with the roto-mixer at the other end of the lab.

Add 250 µl Buffer P2 to each tube.

Snap the tubes closed and invert them 4-6 times, until the tube is thoroughly mixed and the entire solution turns blue.

Add 350 ul Buffer N3 to each tube.

Snap the tubes closed and invert 4-6 times, until the solution is thouroughly mixed and no longer blue.

Centrifuge on high speed for 10 minutes.

Remove the tubes from the microcentrifuge, being careful not to disturb the white pellet.

Using P-1000 micropipettor set to 850 ul, carefully transfer the supernatant from each centrifuge tube to the corresponding blue spin column.

Centrifuge the spin columns for 30 seconds at maximum speed.

Pour the flow-through from each column into the miniprep waste container.

Pipette 500 ul of Buffer PB onto each spin column.

Centrifuge the spin columns for 30 seconds at maximum speed.

Pour the flow-through from each column into the miniprep waste container.

Pipette 750 ul of Buffer PE onto each spin column

Wait 1-3 minutes.

Centrifuge the spin columns for 30 seconds at maximum speed.

Pour the flow-through from each column into the miniprep waste container.

Return each spin column to its collection tube and centrifuge an additional 1 minute at high speed.

Transfer each spin column to a clean labelled microcentrifuge tube.

Pipette 50 ul of Buffer EB onto the center of each column.

Wait 1-3 minutes.

Centrifuge the spin columns, in their collection tubes, for one minute at maximum speed.

Proceed directly to analyze the samples on the Nanodrop.

If it's not running, start the Nanodrop 2000 software. Select "Nucleic Acids."

Ensure that the Type drop-down box on the right-hand side reads DNA.

Ensure that the Use cuvette box on the left-hand side is off.

Raise the Nanodrop arm.

Squirt a Kimwipe with a little water and gently wipe off both the measurement surfaces (the pedestal and the light aperture.)

Use a dry Kimwipe to gently wipe off both measurement surfaces.

Pipette 1.5 ul of Buffer EB onto the pedestal.

Gently lower the Nanodrop arm.

Click the Blank button. Wait a few seconds for the instrument to blank.

Gently wipe off both measurement surfaces.

Pipette 1.5 ul of your sample onto the pedestal.

Lower the Nanodrop arm.

Click the Measure button.

Gently wipe off both measurement surfaces.

Repeat steps 11-15 for each sample.

Squirt a little water on a dry Kimwipe and wipe off both measurement surfaces.

Use a dry Kimwipe to wipe off both measurement surfaces.

Using Benchling, choose a restriction enzyme that meets the following criteria:

Record your enzyme choice on the plasmid's Description page.

Benchling will tell you the enzyme's buffer compatibility and active temperature. Record the buffer in which the enzyme is most active.

Retrieve the minipreps and the appropriate 10X buffer concentrate from the freezer. Thaw on the benchtop or in your fingers.

Label the PCR tubes with your initials and an incrementing number.

Vortex the minipreps and the 10X buffer concentrate briefly, then pulse down in the microfuge.

For each miniprep, set up a PCR tube containing the following in order:

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

Incubate at the appropriate temperature for at least 1 hour and more more than 16 hours.

Stop the reaction by adding 2 ul of 6X NEB purple gel loading dye to each reacti on.

Flick the strip tubes a few times to mix the reaction, then pulse down in the strip tube microfuge.

Proceed to gel electrophoresis.

Check to see if there is a gel waiting in the fridge.

Determine how much molten agarose you'll need.

Measure out the appropriate amount of TAE into a glass bottle or flask.

Add 5 µl SYBRSafe (0.5X ; stock solution is 10000X) per 100 ml of the solution and mix well.

Add UltraPure agarose to a final concentration of 1% (mass / volume)

Swirl the bottle or flask to distribute the agarose.

Heat the solution in the microwave with frequent stirring to dissolve the agarose homogenously. ~30 seconds/100ml solution

Let sit until cool enough to handle.

Set up the gel tray in the casting stand. Make sure the rubber gaskets are flat up against the edges of the casting tray.

Set up the gel combs to form the wells.

Pour the molten agarose into the casting tray.

Wait 30 mintues for the gel to solidify.

Use immediately, or place in a plastic zip-lock baggie with a little 1X TAE and store at 4°C.

If cell culture is in the incubator, remove and check under the microscope for confluence: under 20X magnification, should cover the bottom of the plate

Prepare the hood: fill in the sign-in sheet, turn on the regular light, wipe down with ethanol, and wipe everything down with ethanol before putting it under the hood

Put a glass tip on the vacuum tube. To turn on the vacuum, there is a yellow knob on the left. "On" means the knob is facing the hose.

With a plastic tip on the glass pipette, vacuum the media off the cell plate.

Quickly and gently use pipetter to add 3mL of trypsin to the plate and let sit 3-4 min

Add 10mL of media: pipette up and down to mix. put into 50mL conical tube

Centrifuge (with balance!) at 2300rpm for 4 min.

Vacuum the media off the top and resuspend cells in 10 mL of media

Make the split: there should be 14mL of liquid in the culture dish. a split refers to what amount of the 10mL cell culture is put into the culture dish

Label plates with the cell type, initials, date, and the date of the next splitting (if 1:10, add 3 days, if 1:5, add 2 days, if 1:2, add 1 day). Also put on passage number (how many times the cells have been split.)

Swish gently to mix, put in incubator

Clean up. Suck up any media spilled, or any leftovers in the containers. if the remainder of the media is not enough to use, bleach it. Glass tips go in the sharps. Close the hood, turn the UV on, sign out. Take out the garbage, no matter how much/little there is.

If your samples are frozen, thaw them completely, flick or vortex to mix, then pulse down in the microfuge.

If your samples are not already in loading dye, mix them 1:6 with 6X NEB Purple Loading Dye

Program the voltage on the power supply. For the small gel box, use 100V; for the large gel box, use 150V.

Program the time on the power supply. For a small analytical gel, set the timer for 30 minutes.

Attach the leads to the gel box cover. Make sure that the red (positive) lead is attached to the side of the box farthest away from the wells in the gel.

If the gel box has not been used previously that day, empty it and rinse it out with DI water.

Place the gel in the gel box.

Pour TAE into the gel box until it just barely covers the gel.

Check the wells to see that they are free of bubbles. If there are bubbles, blow them out by pipetting 100 µl of TAE from the gel box into the well.

Load your samples. For large combs, load 10 µl; for small combs, load 5 µl.

Load the ladder in the last lane. Load 1/2 the volume of your samples: for large combs, load 5 µl, for small combs, load 2.5 µl.

Place the lid on the gel box. Make sure it is seated on the brass contacts.

Press the Start or Run button on the power supply.

Make sure the power supply doesn't complain about an open circuit. If it does, re-seat the gel box lid and press Run again.

Double-check that there are bubbles forming on the platinum wires at either end of the gel box.

Double-check that the red (positive) lead is on the side farthest from the wells. Remember, Run to Red.

Run until the pink band is 2/3 to 3/4 of the way down the gel. For small analytical gels, this should take 30 minutes. For larger gels, start at 45 minutes and check regularly!

If the power supply is still running, press the Stop button.

Lift the lid off of the gel box. Lift the lid straight up. If you try to "hinge" it up, the lid will break.

Transfer the gel to the GelDoc.

If it's not running, start QuantityOne from the toolbar.

Press the Epifluorescent Illumination button on the GelDoc. Check the gel's position, zoom and focus.

Close the GelDoc door. Click Auto Expose.

In the File menu, select, Export as JPEG.... Save your gel to the iGEM folder on the desktop.

Open Benchling. Log in, and copy the gel to the Description page for the plasmid you're building.

Discard the gel in the biowaste box. Wipe down the gel doc with a little water and a paper towel or Kimwipe.

Immediately, before you forget what's where, annotate the gel.