The duration of the PCR reaction varies depending on the length of the specific target fragment. For segments of target DNA with less than 1000 base pairs (bp), the DNA polymerase needs to operate for 40 seconds. For the remaining segments which are approximately 6000 bp, the DNA polymerase needs to work for 5 minutes. The entire process is set up as follows:
Add absolute ethanol to Buffer PW as indicated on the bottle.
The volume ratio is as follows:
Place the reaction tube in the PCR machine and let it react at 50℃ for 30 to 60 minutes. After the reaction is complete, the product needs to be placed on ice immediately to cool. Process "Bacterial Conversion" subsequently.
Obtain seed solution: On an LB solid agar plate (100mg/L K antiseptic), inoculate and cultivate for 16-18 hours. Select single colonies with good growth and perform single cloning into 5ml liquid TB medium then cultivate at 37℃, 220rpm shaking incubator for 12 hours. In a 250 mL conical flask, inoculate 50 mL of TB medium with bacterial solution corresponding to 3% of the medium volume (100mg/L K antiseptic), cultivate at 37℃, 220rpm shaking incubator.
Measure the OD value of the shaking-cultured bacterial solution once every hour (add 0.02% volume of defoamer). When OD = 4 (note: dilute according to the range of the instrument), start induction. Add xxx% IPTG (used as experimental variable), induce for xxx hours under same cultivating conditions.
Centrifuge to collect the bacteria. Prepare for ultrasonic disruption and protein electrophoresis detection.
After induction, centrifuge the fermentation broth to collect the cells. Resuspend the pellet in an appropriate amount of protein loading buffer, incubate in a water bath at 100°C for 10 min, and then centrifuge to obtain the supernatant for loading. Pipette the samples into the wells of the protein gel. Set the electrophoresis apparatus at 80 V; once the samples pass the interface between the stacking gel and separating gel, increase the voltage to 120 V. Continue electrophoresis for approximately 45 min until the dye front is about 1 cm from the bottom of the gel, then stop the run.
Place the precipitated cells in an ice bath beaker. Resuspend the precipitated cells in pre-cooled lysis buffer (e.g., 20 mM Tris-HCl, pH 8.0). Extract part of bacterial suspension for ultrasonication and leave the rest for later use. Adjust the ultrasonic probe to a distance of approximately 1 cm from the bottom of the tube. Set the ultrasonic cell disruptor to the following parameters: 25% power, 3s ON, 17s OFF. Run for 20 minutes. Ensure that no significant noise or screeching sound occurs after initiation.
Add the protein loading buffer, and incubate in a boiling water bath for 10 minutes before loading. Set the electrophoresis apparatus to 80V. When the bands enter the separation gel, increase the voltage to 120V.
Prepare a 12% resolving gel and a 4% stacking gel using the Laemmli system.
Mix each protein sample with 5× SDS loading buffer in a 4:1 ratio. Boil the mixture at 100 °C for 10 minutes to denature proteins, then briefly centrifuge to collect the condensate.
Assemble the gel in the electrophoresis tank and fill the inner and outer chambers with 1× Tris-Glycine-SDS running buffer, ensuring no leaks.
Carefully remove the comb and rinse the wells with buffer. Load 5--20 µL of each denatured protein sample into individual wells along with a molecular weight marker.
Run the gel at 80 V through the stacking gel until the dye front reaches the resolving gel, then increase the voltage to 120 V. Continue until the dye front is about 1 cm from the gel's bottom (about 45--60 minutes).
Remove the gel from the glass plates and immerse it in Coomassie Brilliant Blue R-250 staining solution (0.1% dye in 50% methanol and 10% acetic acid). Gently shake for 60 minutes.
Replace the staining solution with destaining buffer (10% methanol, 10% acetic acid). Gently agitate until the gel background becomes clear and the protein bands are sharply visible.
Rinse the gel briefly with water to remove residual destaining solution, then capture images of the protein bands using a gel imaging system for record and analysis.