To use the DNA in the Distribution Kit, follow these instructions:
Note: There is an estimated 2-3ng of DNA in each well, following this protocol, assume that you are transforming with 200-300pg/µL
With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells.
Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.
Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
Use the resulting culture to miniprep the DNA AND make your own glycerol stock.
Transformation - Double Heat Shock
Method
Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
Label 1.5 ml tubes with part name or well location.
Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.
Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.
Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
Close 1.5 ml tubes, incubate on ice for 3 h: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
Heat shock tubes at 42°C for 45 sec: Timing is critical.
Incubate on ice for 2 min: Return transformation tubes to ice bucket.
Heat shock tubes again at 42°C for 45 sec: Timing is critical.
Pipette 250 µL LB media to each transformation
Incubate at 37°C for 1 hours, shaking at 170 rpm
Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates: Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
Spin down cells at 6800 g for 3 mins and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
Count colonies for control transformation: Count colonies on the 100 μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.
Transformation - Heat Shock
Method
Resuspend DNA in selected wells in the Distribution Kit with 10 µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
Label 1.5 ml tubes with part name or well location.
Thaw competent cells on ice: This may take 10-15 min. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
Pipette 50 µl of competent cells into 1.5 ml tube: 50 µl in a 1.5 ml tube per transformation. Keep all tubes on ice.
Pipette 1 µl of resuspended DNA (10 ng- 1 µg) into 1.5 ml tube: Gently pipette up and down a few times. Keep all tubes on ice.
Pipette 1 µl of control DNA into 2 ml tube: Pipette 1 µl of 10 pg/µl control into your control transformation.
Gently pipette up and down a few times. Keep all tubes on ice.
Close 1.5 ml tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
Heat shock tubes at 42°C for 45 sec: Timing is critical.
Incubate on ice for 2 min: Return transformation tubes to ice bucket.
Pipette 250 µL LB media to each transformation.
Incubate at 37°C for 1 hour, shaking at 170 rpm.
Pipette 100 µL of each transformation onto agar w/ antibiotic petri plates (see protocol XXX): Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
Spin down cells at 6800 g for 3 mins and discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads.
immediately. This increases the chance of getting colonies from lower concentration DNA samples.
Incubate transformations overnight (14-18 h) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
Agar with chloramphenicol
Method
Pour 15 ml cold liquid agar in a tube. One tube equals one agar plate.
Pipette 15 µl chloramphenicol to the same tube. If more or less agar is used take 1 µl chloramphenicol per ml agar.
Pour the agar solution in to a petri dish. Spread it out evenly. Let dry with lids almost covering the bottom plates.
Apply cells after directions in transformation protocol found here XXX.
Heat shock competent cells, BL21 (DE3)
Method
Take 25 ml LB medium. Put it in a Eppendorf 50 ml tube
Pipette 50 µl cells to the LB medium. In this case BL21 (DE3) from E.coli
Incubate at 37°C, shaking at 170 rpm. For 4-6 hours.
Thaw cells on ice. For 10 mins (keep cold from now on).
Collect the cells by centrifugation. For 5 mins at 1000 rfc
Decant supernatant. Gently resuspend on 10 ml cold 0.1 M CaCl (cells are susceptible to mechanical disruption, so treat them nicely).
Incubate on ice for 20-30 mins
Repeat step 9, but with 2000 rfc.
Discard supernatant. Then gently resuspend with 5 ml cold 0.1 M CaCl2 + 15% Glycerol
Dispense in microtubes (300 μL/tube). Freeze in -80°C.
GenElute™ Plasmid Miniprep Kit
The protocol and material used for plasmid purification was from Sigma-Aldrich
Centrifuge 5 ml of overnight grown competent cells for 1 min at 1200 rcf. Discard the supernatant.
Resuspend cells. Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous.
Lyse cells. Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous. Do not vortex. Do not allow the lysis reaction to exceed 5 minutes.
Neutralize. Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifugation at 12,000 g for 10 min.
Prepare column. Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to miniprep column and centrifuge at 12,000 g for 1 min.
Discard the flow-through liquid.
Load cleared lysate. Transfer the cleared lysate from step 4 to the column prepared in step 5 and centrifuge at 12,000 g for 1 min. Discard the flow-through liquid.
Wash column. Add 750 µl of the diluted Wash Solution to the column. Centrifuge at 12,000 g for 1 min. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 min without any additional Wash Solution to remove excess ethanol. Discard the flow-through liquid.
Elute DNA. Transfer the column to a fresh collection tube. Add 50 µl molecular biology reagent water to the column. Centrifuge at 12,000 g for 1 min. The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C.
