Plasmid Resuspension l
Resuspend ordered plasmid (construct) in a 0.1mM Tris, 0.1uM EDTA buffer solution. Ensure circularization with transformation.
Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins). Remove agar plates (containing the appropriate antibiotic) from storage at 4°C and let warm up to room temperature. Mix 5 μL of DNA into 20 μL of competent cells in a micro centrifuge tube. Incubate the competent cell/DNA mixture on ice for 20-30 mins. Heat shock each transformation tube by placing the tube into a 42°C water bath for 45 seconds. Put the tubes back on ice for 2 min. Add 250 μL LB to the bacteria and grow in 37°C shaking incubator for an hour. Plate 200 uL and 50 uL on 2 separate plates. Incubate plates at 37°C overnight.
Using a NanoDrop, place an initial blank of d2H2O. Remove the water then add a DNA sample then use the 260/280nm nucleic acid measurement tool to quantify DNA concentration.
Add 10g of Tryptone, 10g of NaCl, 5g of Yeast Extract to just under 1L of d2H2O. Take the solution and obtain a pH of 7.5. Autoclave to obtain LB media or add 5g of agar to 500 mL then autoclave to obtain agar plates.
Overnight Culture (Colony pick)
Identify a colony on an LB + Agar plate and with a small pipette tip place the tip into the colony then removing it without damaging the agar in and around the colony. Place the pipet tip into a 5 mL LB media tube. If needed add appropriate antibiotic to the solution. Amp was used for our construct. Incubate tubes at 37°C overnight but ensure that it doesn’t stay too long or the antibiotic can become deactivated leading to contamination.
In a 1.5 mL tube add the following components and make alterations based on DNA concentrations so that the tube can have a final volume of 30 uL. Add approximately 1 ug of DNA, 1 uL of each Restriction Enzyme, 3 uL of 10x Cut buffer. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Then if using this DNA right away deactivate the enzymes by bringing the temperature up to 70°C for 15 minutes or longer.
Combine the following in a PCR or small micro centrifuge tube: 25ng Vector DNA, 75ng Insert DNA, Ligase Buffer, 1μL T4 DNA Ligase and d2H2O to total it to 10 or 15 uL. Incubate at room temperature for 2hr, or overnight. Proceed with bacterial transformation.