PCR Detection of Genetically Modified Foods: Electrophoresis (Part III:…
PCR Detection of Genetically Modified Foods: Electrophoresis
Part 1: Pour Agarose Gel
Choose 8-well comb, can also use 16.
Put comb far from red so you get a good run.
Place the black barriers to keep the agarose inside the tray.
Fill tray with molten agarose until 2/3 full.
Lift top of gel box and place tray. It is clear and looks like a U.
Wait 20 minutes for gel to set.
Wear gloves for protection.
Part II: Prepare Samples
Add 5 uL of loading dye to each PCR sample.
Vortex each sample.
Vortex loading dye.
Write down which sample goes in each well. DNA ladder belongs in first well.
Obtain DNA ladder sample that contains loading dye.
Part III: Load DNA Samples into Gel
Remove comb and wipe it with a moistened lab tissue and put it back in front of lab.
Move gel box near power supply you plan to use.
Pour TBE buffer into gel box until it just covers surface of gel. Don't want any gel sticking up above top of buffer.
Load 10 uL of DNA ladder to each well of gel.
Remove barriers and wipe them with a wet tissue and dry tissue.
Move any air bubbles in well out with a pipette tip.
Once 20 minutes have elapsed since the gel was poured, continue.
Don't hit second stop on micropipettor when loading well.
Load 15 uL of each remaining sample into appropriate wells of your gel.
Tell them if you screw up and lose sample.
Part IV: Running Gel
Set to 115-120 volts.
Look for bubbles forming near electrodes at base of gel box and make sure current running through circuit is greater than 0.
Look out for if the machine is already on or if somebody is already using it to run a sample.
Let it run for 40-60 minutes, or until faster loading dye band has traveled 2/3 of the length of the gel.
Put lid back on box.
Record voltage you used and the amount of time you let it run.
Part V: Photographing the Gel
Align photo so that wells are at top. Use permanent marker to label lanes of gel.
Use numbers or symbols for each lane and make a legend to explain what each number represents.
Only dispose of gel in biohazard box once you are sure that both photographs have come out okay.
Put date and initials on photograph.
Pour buffer back into stock container marked TBE.
Remove gel and put it in weigh boat. Push exposed edge of gel with finger to make it slide off tray and into weigh boat.
Label edge of plastic weigh boat with your name.
Turn off power and unplug the gel box. Then remove the top of the gel box.
Use results to complete lab assignment.