How to Cut DNA from an Agarose Gel

hi everybody welcome back to synthetic biology one today I probably look a little bit funny and that is because we are using our special DNA cam that is equipped with optical filters that will allow you at home to visualize the fragments of fluorescently labeled DNA that we're going to be working with today I will show you how to cut bands of DNA out of an agarose gel for purposes of purification okay so let's get started I've got everything I need right here so I'm wearing my my gloves my nitrile gloves I've got my my agarose gel here I've got 1.5 mil eppendorf tubes to collect the band of the gel that I cut out I have my disposable scalpels that I'm going to be using to perform the actual cutting and I've got ethanol and kin wipes to clean up after I'm done here is the gel that we're going to be working with today I will cut the bands from this gel and we'll go through the protocol two times first in the natural light so that you can see the manipulations and then second using the fluorescent light so that you can hopefully see the DNA that we're actually cutting so here's our basic strategy for cutting a band out of an agarose gel first I want to identify the lane of the gel where I expect to find my band then I will cut very precisely on one side of the gel and separate away the unwanted fragment then I will do the same thing on the other side of my band until I have left only the lane of the gel that contains my DNA fragment by the way in a lot of labs they don't like you to cut directly on the gel box imager and that's because you can scratch the surface which over time will make it ugly and less functional but today my lab manager is not looking so I am just going to go ahead and do it once you've isolated the band or the lane rather that contains your gel we'll make one more cut just above our band in the gel here the key is going to be to come down directly vertically with the scalpel and not in not at an angle in order to make a nice straight cut in our in our gel and then one more time just below the DNA band that we're trying to isolate so we're looking for the thinnest possible slice of gel that still contains our DNA fragment something about the width of a coin right and not not like a quarter not like an American coin something a little thicker than that like a like a European coin like a serious like a real a real coin then when we're done we'll take a 1.5 mil tube scoop up the band and take it away it's just that simple now I will clean up this gel for the next person and for the second time through the procedure we will enter fluorescence mode so overhead lights off illumination tool on and there we have it so I can see because I'm wearing these these orange glasses that filter out all of the blue light that's produced by this illumination table but not the green light that's produced by the fluorescently labeled DNA I can see these bands perfectly as I hope you can to at home so we've got over here the DNA ladder so this is a series of DNA fragments of known sizes that we can compare to our fragment in order to determine the size of our DNA fragment and over here I see two brightly lit well-defined fragments of DNA so the the the larger one is is here closer to me on this gel and the the smaller one is below further away from me so first cut just to one side of the band that I'm trying to isolate and then we remove the unwanted piece of the gel so now that I've separated the gel it's very important that I remember which of the two bands that I want because without the ladder there for Association I won't be able to tell anymore which band is larger and which band is smaller second cut just to the right third cut just above the DNA fragment that I'm isolating and fourth cut just below the fragment that I've isolating and there you have it so this should be this is about a 100 milligram slice of gel thick enough to contain all of the DNA in our band but not too thick which means it will be easy for us to purify this agarose to remove it and separate it from our DNA later on in a protocol now I take a 1.5 mil up in Dorf tube very carefully scoop up my oops scoop up my band of DNA with the scalpel very carefully since it's so sharp well drop it into the tube and there you go a perfectly isolated band of DNA cut from an agarose gel and ready for downstream purification and use in the application of your choice that's all for today until next time stay bright

Channel: Synthetic Biology One

Views: 51,322

Rating: 4.8499279 out of 5

Keywords: Synthetic, Biology

Id: QHYWRd2M-gw

Length: 7min 48sec (468 seconds)

Published: Mon Mar 13 2017