Big DNA QC on Pulse Field Gels

  • Decide the size range of DNA molecules for separation. You need to determine the smallest and largest molecules of interest that will appear on your gel.

  • Use the ‘auto algorithm’ function on the CHEF gel mapper to determine the conditions for the run. In addition to the run time, switching time, initial voltage etc., the algorithm will also tell you the percentage gel to prepare as well as the buffer. Most of the time these parameters will be 0.5X TBE and 1% agarose gel (made up in 0.5X TBE), but it is always good to double check prior to pouring your gel.

  • Prepare 2L of running buffer (e.g.0. 5X TBE). (Note that you can buy 10X TBE at the store in Smilow.)

  • Using low melting point agarose or CHEF gel agarose (from Biorad), prepare 100mL of the melted gel solution. For example, if you want to make a 1% gel, then use 1g of agarose. To avoid clumps of agar that may not completely melt, it is advisable to first add the 100ml of buffer to an Erlenmeyer flask containing a stir bar. With the stir bar spinning on a stir plate, gently tap the agarose into the Erlenmeyer a little at a time until it is all in the solution and stirring around.

  • Microwave until the gel is completely melted. By bringing the gel to a boil 3 sequential times you can ensure all of the tiny bits of agarose are completely melted.

  • Add back ~5-7 ml of water to account for the volume lost during boiling and swirl thoroughly.

  • Cool the gel for about 20 minutes on the benchtop until it is still warm to the touch and no longer steaming.

  • Pour the gel into the assembled casting tray.

  • Let the gel stand for at least an hour to completely solidify.

  • Make sure the tubing is connected correctly and that the tube for draining the buffer tank is not connected. Pour the remaining 1.9L of buffer into the gel apparatus.

  • Turn on the instruments in the following order (note that if you do this in the opposite order, residual liquid inside the chiller could freeze and block the flow of buffer; if this happens you will have to turn off the chiller and wait until the system warms up and the liquid melts):

  • Power box (two switches)

  • Pump (turn pump switch up to 65 or 70)

  • Chiller (set to 14C, but change to appropriate temp based on run conditions set by auto algorithm)

  • While gel sets, let buffer chill to 14C with circulation.

  • When the gel is set, remove it from the casting tray and carefully wipe off the bottom of the black plastic part underneath the gel using a paper towel. This part will go into the buffer tank and should be free of loose agarose, which could clog up the tubing.

  • Load your samples that have already been mixed with loading dye.

  • Run your program.

  • When the program is complete, transfer the gel (and black supporting part) to a glass pyrex dish with 300mL water + 15uL EtBr (5mg/ml). Alternatively, you can stain your gel with sybr gold or other non-toxic and possibly more sensitive reagents.

  • Incubate gel with very gentle rocking for 30 minutes

  • Replace EtBr water with fresh water and wash for 15 minutes

  • Image gel.

  • Replace water wash with fresh water and incubate gel with gentle rocking for 15 minutes.

  • Image gel

  • Repeat steps 20 and 21 until you are happy with the gel image.

  • Clean buffer tank with 2L water repeatedly until water wash no longer has residual gel chunks.

Notes:

  • If you want to run FIGE, you need to select 2kb-50kb when you program the instrument using the auto-algorithm function.

  • You need to consider up front what ladder you plan to use. We have two (monocut (1-50kb, N3019S) and lambda phage (50kb ladder pattern, N0341S) and they are stored in the -20C freezer inside the automation suite (adjacent to the automate -80C freezer).

  • NEVER re-use buffer. Always change between each run.

  • For further information, please check the CHEF mapper manual