915px Lambda Red, PCR to get the required insertion product (zeocin in this example)

Procedure

Part A) PCR to get the required product.

• We designed primers flanking the region of interest (the zeocin cassette), and added 30 to 50 base pairs of homology matching the ECNR2 genome flanking the rpoZ gene. The lengths of the annealing regions of the primers are chosen so that they have a similar (within 1°C ) melting temperature for the PCR reaction.
• The PCR reaction is done with the above primers, and a template of a small amount of liquid culture (diluted 30 times) of the omega knockout bacteria hybrid selection strain.
• The product is run on a gel, and then either gel extracted (if there are side products) or PCR purified (if the reaction ran clear) and eluted in molecular grade water, not EB, since the ions and salts would otherwise cause electrical arcing during electroporation

Part B) Lambda-red recombination

• 1) Activate lambda red through temperature sensitive activation. For the ECNR2 strain, this involved taking the culture that’s grown in LB, at 30°C at around midlog, and placing it in a water bath at 42°C for 15 min to activate the lambda red machinery.
• 2) Add 1-1.5 mL of mid-log cells in culture to a 1.5 mL microcentrifuge tube. Spin in the centrifuge for 1 minute at 15000rpm (max speed)
• 3) (All steps need to be done on ice and the pipette tips, the cuvettes, and the molecular grade water must be kept cold at all times.)
• 4) Decant the liquid at the top, and use a pipette to remove as much of the liquid as possible without disturbing the bacterial pellet. Change tips. Wash cells with 1 mL of cold water, pipette up and down to mix them well, and spin in centrifuge for 1 minute at max speed.
• 5) Repeat the wash step.
• 6) Tip: When you are removing the liquid after spinning down, it helps to hold the tube so that the bacterial pellet is at the top and then use the pipette to draw out the liquid right below it. This is done so that the salts from the culture media can be thoroughly washed away, as they could interfere with the use of the electroporator from Step 10 (and onwards).
• 7) Resuspend cells with 50 μL of cold water.
• 8) Add 50-200 ng of insertion construct to the microcentrifuge tube with the bacterial pellet. This could also could be accomplished by taking a separate microcentrifuge tube, and adding enough of the insertion construct to almost 50 μL of cold water in an earlier step, so that these steps can be accomplished more quickly. Since the PCR product(insertion construct) from part A was at a concentration of about 70ng/μL to about 200ng/μL for the different times we used this method, we usually added 1-2μL of the construct to about 48-49μL of water in a microcentrifuge tube and placed it on ice before we began part B from step 2. It helps to reduce the amount of time the bacteria are suspended in water since it can lyse the cells because the surrounding solution is hypotonic to the bacterial cytoplasm.
• 9) Add the 50 μL solution to the cells, pipette up and down to mix them well, and then place in a chilled cuvette.
• 10) Set the electroporation machine to Eco1, 1.8 volts. A time constant of 4.8ms is a good number. You can also run a 'blank' to figure out the optimum time constant. A blank is run by filling a cuvette with 50 μL of water, which is put in the electroporator and pulsed, and its time constant is taken as as the standard. We want a time constant as close to that for water as possible, because pure water has no salts.
• 11) Before placing the cuvette in the electroporator, make sure that all water is wiped off of cuvette, so that it is dry, or it can interfere with electrical contact.
• 12) After electroporation immediately place 1 mL of LB in cuvette, pipette up and down to mix the cells in the LB and resuspend them.
• 13) Then place that 1mL of bacteria in LB into 2 mL of LB in a culture tube and allow them to grow for 2 to 3 hours in correct temperature (30°C for ECNR2).
• 14) After three hours, plate out culture in different concentrations of bacteria on specific plates (in this case, of zeocin, since that was in the construct, and it allows us to select for the lambda red insertion).
• 15) Grow up the cultures on the plates overnight, and use a colony to do PCR or sequencing to check for the insert via its sequence, or the band size on the gel.