Electrophoresis Gel Procedure<\/p>\n
Materials:
\nTAE buffer (In transparent plastic bottle)
\nErlenmeyer flask with cap (glass petri dish)
\nWeigh paper
\nPCR Grade Agarose (1% agarose in buffer)
\nElectrophoresis machine
\n2 gel combs<\/p>\n
Procedure:
\n\u2022\tWeigh 0.7 grams of agarose using the blue plastic spoon in the agarose jar, and put this in the flask.
\n\u2022\tMeasure 70 mL of TAE buffer and pour into the flask
\n\u2022\tHeat the flask with cap in the microwave at 2 minutes until you hear a tinkling sound \u2013 at this point take it out and swirl. Use the green PPE glove.
\n\u2022\tRepeat microwaving in increments until you hear the tinkling sounds. Keep repeating until the agarose is completely dissolved (it will not look like there are clear granules in the liquid).
\n\u2022\tLet the flask cool after microwaving (you may run water over the flask to cool it faster). It should cool to about 50 degrees C before you pour it. The flask shouldn\u2019t be painful to the touch.
\n\u2022\tEnsure that the gel casting tray is clean and dry. It may be cleaned with DI water if dirty. Never use bleach to clean the acrylic.
\n\u2022\tEnsure that the gel container is oriented so that the current will run from black to red.
\n\u2022\tCheck that the gel container is level using the rounded level.
\n\u2022\tEnsure that the gel casting tray is oriented so that the seals are against the walls of the gel container.
\n\u2022\tPour the gel from the flask into the casting tray. Use a pipette tip to remove any bubbles.
\n\u2022\tInclude 2 gel combs in the notches in the tray. Note: the combs have different volume sizes and amount of wells on each side, so be sure to pick the well volume and number of wells you want.
\n\u2022\tThe gel takes 1.5 to 2 hours to fully solidify. It can be checked intermittently during this time, and will turn opaque when solid and dry.
\n\u2022\tTurn the gel casting tray so that the well contents will flow from black to red. If oriented from red to black, the bands will just fall off of the gel into the buffer. Pour buffer so that it is touching the electrodes.
\n\u2022\tOnce buffer is covering everything, the combs may be taken out. Be careful not to rip the gel. Pull the combs directly upwards. <\/p>\n
Filling Wells<\/p>\n
Materials:
\nSYBR green (In freezer)
\nLoading dye
\nLadder 100 BP (fridge)
\nApproximately 6 inches of parafilm
\n10 uL pipette and tips<\/p>\n
Procedure:
\n\u2022\tBeforehand, mix 300 uL SYBR green with 600 uL loading dye and keep in freezer.
\n\u2022\tCentrifuge the PCR products and then put in a frozen rack. Centrifuge SYBR green mixture and put in the larger frozen rack wrapped in aluminum foil. It is light sensitive.
\n\u2022\tAdd SYBR green mixture in dots on the parafilm, with 2 uL of the mixture per dot. You will need the amount of dots for your number of samples plus 1 for the ladder. (8 dots for 5 beetles, a negative and positive control, and the ladder).
\n\u2022\tAdd 6-8 uL of ladder in the first dot only, and pull the liquid up and down in the pipette tip to mix. Note: if electrophoresing two rows of wells, it can be helpful to put a ladder in the first well of the second row as well as the first.
\n\u2022\tAdd 6 uL of each PCR product in each of the other dots and mix up and down in the pipette tip (It is important at this point to make sure you know what order your samples are in).
\n\u2022\tSet the pipette to 9.5 uL and pull the first dot up with the pipette. Be careful not to introduce air bubbles. Add the liquid to the first well as vertically as possible, and being careful not to touch the gel. Don\u2019t press the pipette button down the second half of the way so that air bubbles are not introduced. It is better to leave a very small amount of PCR product in the tip than to introduce bubbles.
\n\u2022\tRepeat this with each of the dots into each of the wells.
\n\u2022\tSlide the lid onto the gel container.
\n\u2022\tPlug the red cord into the red outlet, and the black cord into the black outlet on the machine.
\n\u2022\tTurn the machine on, and put on 100 volts for 45 minutes.
\n\u2022\tTake a photo of the gel within 10 minutes after it finishes. <\/p>\n
Gel Photos Procedure:
\n\u2022\tTake the gel casting tray out of the gel container and place it into the tray of the Enduro machine. Make sure everything on the machine is closed, because it emits UV light to take the photo.
\n\u2022\tOpen the Enduro GDS software on the computer, and click capture image, then click illuminate. This should make the gel with bands visible. You can adjust the illumination to make the bands more visible. Once done adjusting, you can take a photo.
\n\u2022\tTake the gel casting tray out of the machine, making sure the UV is off first.
\n\u2022\tGel can be thrown away, and the gel casting tray can be cleaned with DI water. Wipe the Enduro tray with DI water as well. <\/p>\n
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.<\/p>\n
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.<\/p>\n
For loading directly to the wells in the gel with your sample<\/p>\n
From www.lonza.com: SYBR\u00ae 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\u00ae Green I Stain in high-quality anhydrous DMSO. The 1:100 dilution can be stored in the freezer and reused. Add 1 \u03bcl of this dilution to 9 \u03bcl-10 \u03bcl of your sample before loading.<\/p>\n
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:<\/p>\n
For mastermix, combine 300uL of 100x SYBR and 600uL of Dye.<\/p>\n
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.<\/p>\n
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.<\/p>\n
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.<\/p>\n
A cheap and possibly mutagenic alternative to SYBR Green is 1:1000X Ethidium Bromide added directly to the agarose after cooling. WEAR GLOVES!<\/i>
\nLoad DNA ladder in the last well.<\/p>\n3D Printed gel combs<\/h2>\n
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<\/a>.<\/p>\n 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.<\/p>\n