Protocols

After running a PCR, we need to determine if the PCR was successful before moving on.

Step 1: Place prepared gel into gel apparatus, and then fill with 1X TAE until liquid covers the top of the gel

Step 2: Prepare the samples by aliquoting 5 uL of sample with 1 uL of loading dye

Note: If PCR was prepared using OneTaq polymerase, this step is unnecessary

Step 3: Add 5uL of the prepared samples to each slot, and add 5 uL of DNA ladder

Step 4: Put the lid of the apparatus on and set the power supply to 120V for 45 minutes. Note: Make sure that the Gel slots align with the negative end of the gel box

Step 5: Image using BioRad gel imager

To make a Gel:

Step 1: Add 1g of Agarose powder to 100 mL of 1X TAE buffer

Step 2: Microwave until combined, and then add 10 uL of Syber Safe Stain

Step 3: Pour into prepared molds, and then place comb. Wait a few minutes until cooled to use

Step 1: Prepare LB Agar media. Mix LB Agar (Miller) power with DI water. Sigma-Aldrich LB Broth with Agar powder was used for plates

Note: 40g of LB agar powder per 1 liter of DI water

Step 2: Sterilize and dissolve LB-Agar mixture by microwaving in 30 second increments until powder is completely dissolved, swirling container between each increment until clear.

Note: Autoclaving can also be used for this step: 20 min at 15 psi (1.05 kg/cm2) on liquid cycle

The following steps should be done in a sterile Biosafety Cabinet (BSC):

Step 3: Allow mixture to cool for 5 minutes and add sterile antibiotic, swirl container to mix:

Step 4: Before LB-Agar mixture is completely cool pipette 12mL of mixture into each plate. Allow plates to set without lids on to avoid condensation 

Plates can be stored at 4ºC until use