ProceduresPCR ReactionCollect all equipments required for the experiment (CS Tube, A Tube, B Tube, C Tube, D Tube and MMP Tube) and label them accordingly with our group name (RJ). Using a pipette, pipette 20 microlitre of MMP and add it into each tube (CS, A, B, C, D) respectively. Mix the contents well. Use a new pipette tip after each tube has had MMP added and mixed in well. During this procedure, the other remaining 4 tubes have to be placed onto a rack to prevent any mix up.
After which, the tubes have to be placed into a thermal cycler for 35 cycles that would take about 3 hours. Note the position at which your tubes have been placed. Gel ElectrophoresisTo prepare the agar, add in 3 litres of TAE electrophoresis buffer with 60ml of concentrate 50x TAE buffer and 2940ml of distilled water. Through heating in the microwave, dissolve 1x TAE electrophoresis buffer with 10.5g of agarose powder.
The microwave heating should be set at 60°C In the meanwhile, set the casting tray with the comb located at 1cm for easy reading. Once the gel is ready, pour 30ml of it into the casting tray and leave it to solidify for 20 minutes. After which, remove the comb from the casting tray that has the gel and subsequently placing the tray into the DNA electrophoresis chamber. Ensure that the chamber is levelled with the table and the wells are facing the cathode (black) end.
Fill the chamber with 1 x TAE running buffer till it touches the maximum line.In order to have a balanced microcentrifuge, place your PCR tubes collected from the thermal cycler into the pulse-spin. Ensure that the liquid is collected at the bottom of the tube at the end of the procedure. Next, pipette 10 microlitre of the loading dye into every PCR tube. Ensure that the liquid is thoroughly mixed by the pipette tip by the action of up and down mix. Followed by placing the tubes in pulse-spin for a quick mix.
The crucial part of this experiment is loading the samples into the wells that would determine your final result. The samples have to be added into the wells in a systematic order as follows:Allele Ladder with orange loading dyeCS DNA with orange loading dyeSuspect A DNA with orange loading dyeSuspect B DNA with orange loading dyeSuspect C DNA with orange loading dyeSuspect D DNA with orange loading dyeOnce the samples have been added into the wells without any spillage, run the gels for 100 volts for 30 minutes or till it reaches 7.5cm (whichever gives a better result)After 30 minutes, remove the gel and place it into the tupperware with 1x Fast Blast Staining Solution for Overnight Staining Protocol. Cover the gel totally with the staining solution. Let it rest for 5 minutes before de-staining it using distilled water to check for the stained gel.Lastly, when you are satisfied with your destaining result, take a photo of it and explain it in your report.