For the small tray, you need 60mL of 1% gel (0.6g of agarose to 60 ml of TAE buffer). Boil over in microwave (about 2 mins). Cool down under cold water, pour into the rig with combs, wait for about 15 min.
For the medium tray, you need 160mL of 1% gel (1.6g of agarose to 160mL of TAE buffer). Boil over in microwave (about 2 mins). Cool down under cold water, pour into the rig with gel combs, wait for about 15 min.
Preparing SYBR Green
For loading directly to the wells in the gel with your sample
From www.lonza.com: SYBR® Green I Stain can be added directly to the loading buffer at a final concentration of 1:1000. First prepare a 1:100 dilution of SYBR® Green I Stain in high-quality anhydrous DMSO. The 1:100 dilution can be stored in the freezer and reused. Add 1 μl of this dilution to 9 μl-10 μl of your sample before loading.
SYBR Green & Dye mastermix
For one sample, combine 0.5uL of 100x SYBR (labeled 1:100X) and 1uL of 6X Loading Dye. Mix that with 5uL of PCR product. Or, more typically, make and store a mastermix:
For mastermix, combine 300uL of 100x SYBR and 600uL of Dye.
Loading the Gel
Prepare ~1.5uL droplets of SYBR & Dye mastermix on parafilm, there are aliquots of this mix prepared by the lab manager in the shared reagent box in the door of the small freezer. Add 5uL of PCR product, mix by sucking up & down with the pipette. Load into slot on gel.
For GreenTaq PCR product (product is green): add 5uL of sample to respective droplets of only SYBR Green, mix by sucking up&down with the pipette. No other loading dye needed.
Run on about 110 Volts for 45 minutes for running about halfway down the gel. Lower time for a shorter distance. If there is not enough liquid in the gel tray, add TAE buffer, just enough to cover the gel.
A cheap and possibly mutagenic alternative to SYBR Green is 1:1000X Ethidium Bromide added directly to the agarose after cooling. WEAR GLOVES!
Load DNA ladder in the last well.
3D Printed gel combs
We have designs for 3D printed gel combs compatible with our gel trays. These combs are designed to match our multichannel pipettes and rest either on top of, or in the slots of, the gel trays. The designs have three pieces: (1) the comb itself, slotted to fit into one of the holder types and with single and dual lane options; (2) an on top of tray holder which allows you to place the comb at any distance down the tray, and (3) an in line holder which allows you to use the standard distance slots in the tray. Links to the 3D printable files can be found below. You may print these through the UF libraries for a low cost as student, faculty, or staff.
PLEASE DO NOT EDIT THESE DESIGNS. To prepare one for printing, simply select the piece you would like to print, choose “Export,” and export just that piece as an .stl file. This can then be uploaded to the printing lab. Do not try to download all three pieces as one file, each file must be a single connected piece. You can submit multiple files at once to have them printed at the same time.