MAKING_LIFE 2014 - 2015

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Restriction enzymes - part 2
posted by sarah on 16 March 2016

Next up is part two of the work with restriction enzymes with Paul Vanouse. The DNA itself is quite light and will generally float up out of the wells within the electrophoresis gel. To avoid this we add 2µl of loading buffer into each of the four vials; the uncut lambda DNA and vials A, B and C which contain the up to now unknown restriction enzyme. The loading buffer serves two purposes; one being to add a blue dye which makes the DNA more visible, and the other is to attach heavy sugar molecules that will serve to make it sink down into the wells. It is also a good idea to centrifuge for a few seconds to make sure that this tiny amount of liquid is all down at the bottom of the tube. The tubes are then warmed at 65º for 5 minutes in the water bath before being put on ice until ready to load.

There is a large gel with 32 wells in two rows of 16 already set up. It would have been interesting to go through the process for making a gel but time restrictions don't allow us to do every step ourselves. This link explains the fairly simple process quite well: In fact, it gives a nice overview to the whole DIY electrophoresis approach. Another important step is to get the bubbles out of the wells, and this can be accomplished simply by rubbing and tapping your gloved fingers across the tops.

Those that have never pipetted DNA into the small wells in a gel before, and those that wanted to brush up on their technique a little, spent a few moments poking around the gel with a pipette tip. Paul emphasised that at this stage of the game sterility was not so important as it's the last part of the process. After loading the first two lanes with pure lambda and a marker sample - which basically serves as a 'reader' against which to compare our own lanes - each group pipetted their 3 samples into 3 lanes and tried to remember which was which. Its good to work quickly as the DNA, even with the loading buffer, tends to begin to float up out of the wells quite soon.

After solving some issues with electrodes, connecting wires and an incessantly beeping power supply the gel was run at 100V for a minimum of 40 minutes. The voltage is proportional to the time it takes to run a gel - more voltage is faster and less voltage is slower.

Paul had earlier provided a text file of the lambda sequence as well as the cutting sites of each of the enzymes. using a basic search function on a text editor we should be able to work out how and where each cuts the lambda and thus identify which is which by analysing our results in the gel. Orkhan also showed some software at Gentle2 from which automatically finds the restriction enzymes and sites. (new sequence > enter sequence > copy /past > name sequence > display settings [eye icon] section "restriction enzymes")


On checking the gel the problems we had in getting it running are evident. Some of the DNA had escaped and the gel had not run for the optimum time. Nevertheless we had well defined ladders for each of the enzymes and Pei showed us how easy it was to work out and visualise the cuts using Sublime Text. A is HIndIII, the 12 cuts in B identify it as PstI and lastly C is ecoRI. The suspects were identified.