This protocol details a typical setup for screening for transformed cells in the process of cloning a gene following a typical cloning procedure. An alkaline phosphotase is use to ensure there is no self ligation of the vector.
α-Complementation - Many shuttle vectors contain an ampicillin resistance gene so only successfully transformed cells may grow on ampicillin containing media. They also contain the β-galactosidase α-fragment. Using functional complementation, the expression of this fragment in cells with the lacZΔM15 mutation allows functional expression of β-galactosidase. Cells expressing this enzyme can hydrolyze the modified sugar X-gal into a blue product, so transformed colonies can be easily identified by their blue color. As the β-galactosidase α-fragment typically lies within the MCS of the lacZ gene, successful DNA insertions into the vector plasmid stops functional expression of the β-galactosidase gene. Thus cells transformed by plasmids containing an MCS insert appear white and uncolored. This protocol also details the use of IPTG, a non-metabolizable galactose analog, to induce expression of the lacZ operon. Both IPTG and X-Gal will be incubated with the LB-Amp plates before inoculation with transformed bacteria.
Alkaline Phosphatase Treatment
Add 1 µl of enzyme and 1.5 µl digested vector to a microfuge tube.
Add ddH2O to bring the total volume to 20 µl.
For a control untreated vector, add 1.5 µl digested vector and enough ddH2O to bring the volume to 20 µl to a microfuge tube.
Incubate the reactions for 1 hour at 37 oC
For both the treated and untreated vector, add 100 µl of DNA binding buffer to the 20 µl samples and vortex.
Transfer solution to Zymo-Spin column placed in a collection tube. Any miniprep spin column will as long as it is usable for the length of dsDNA you are treating.
Centrifuge column at ≥10,000g for 30 seconds and discard flow through.
Add 200 µl Wash buffer and centrifuge at ≥10,000g for 30 seconds. Repeat for a second wash.
Incubate column with 50 µl elution buffer for 1 minute. Move column to a microfuge tube and centrifuge at ≥10,000g for 30 seconds.
Reapply eluate to Spin column and incubate for 1 minute. Spin for 30 seconds at ≥10,000g to elute final product.
Ligation of Insert
Add 50 ng of the vector (treated and untreated) and a 3-fold molar excess of the insert to a microfuge tube.
Add 50 ng treated vector without insert to a microfuge tube for the vector only control.
Add dd H2O to each tube to bring the total volume to 10 µl.
Add 10 µl 2X NEB Quick Ligation Buffer to each tube.
Add 1 µl NEB Quick ligase to each tube. Mix the reaction by pipetting up and down.
Incubate at room temperature for 5 minutes.
Move the reaction tubes to an ice bath.
Transformation of E. coli
Prepare the water bath at 42 oC and the agar plates at 37 oC.
Thaw 3 tubes with competent cells in ice bucket.
Add 2 µl of each different ligation reaction to each tube. Be sure that the added volume is less than 5 µl. Tap the tubes gently to mix.
Incubate the tubes for 30 minutes on ice.
Heat shock the tubes in the water bath for 30 seconds.
Place the tubes back on ice for 2 minutes.
Add 250 µl SOC media to the tubes using aseptic techniques. Incubate the tubes at 37 oC and 225 – 250 rpm on a shaker for 1 hour.
media. Place on the shaker with the sample tubes and incubate for 1 hour.
Add 240 µl IPTG stock and 240 µl X-Gal stock to a microfuge tube. Mix gently.
Add 80 µl IPTG X-Gal mix to 6 LB-Amp plates. Spread the mixes over each plate using aseptic technique and a glass spreader. Incubate the plates at 37 oC for 20 minutes.
Plate 50 µl and 150 µl from each tube onto two separate LB-amp plates, for a total of 6 plates.
Incubate the plates overnight at 37 oC.
The AP treated vectors cannot form a circular plasmid without the presence of an insert, so your treated vector + insert reaction should contain only recombinant circular vector. Bacteria transformed with this reaction mix have a non-functional β-galactosidase, and thus appear white. If any bacteria happen to have innate ampicillin resistance, white colonies will appear on plates streaked with these cells. Untreated vector in still has 5’ phosphates, so the reaction creates both nonrecombinant plamids that recircularized without an insert, as well as recombinant plasmids with the insert. The nonrecombinant plasmids create blue colonies due to alpha complementation restoring β-galactosidase function, while the recombinant plasmids create white colonies due to the disruption of the β-galactosidase gene.