DNA electrophoresis sample loading

the purpose of this video is to point out concerns when loading samples for horizontal DNA electrophoresis the first thing to be concerned about is actually the preparation of your DNA sample you'll want to dilute your DNA sample into a loading buffer typically these are found in 10x or 6x formations and they essentially contain weighting agents which are typically cycle glycerol or sucrose or commonly used and also they will contain a dye or a couple of dyes that will allow you to monitor the electrophoresis process it is important that these are diluted properly otherwise your DNA will actually dissolve into the aqueous buffer versus actually dropping into the wealth and that's the importance of the glycerol or the weighting agent secondly it's important to know approximately how much of your sample you will be loading because you want to make sure that the wells in your gel will hold that amount if you load more into the well it will spill over into the other wells and contaminate you and give you possibly false results the first two examples I want to show you are normal loading notice I put the tip down into the well when I depress the plunger the sample will drop down to the bottom as I pull away you'll also notice the small stream if this is a word you just hold your tip down there longer so the sample can drop down otherwise it's usually no big deal when you're loading a sample the next couple examples are your classic air bubbles the first one is the air bubble at the tip this is usually no problem just before you put it into the buffer or to the electorate electrophoresis buffer just to press your plunger to get rid of it the second one is air bubbles within the tip if you see this be aware of it before you try to load it put it back into your tube draw it back up without air bubbles air bubbles in the tip are going to cause dispersion of your sample and possibly lead to some of the loss of your sample here is the classic shoving your tip through the bottom of the gel just be aware of where your tip is as you can see down at the bottom the sample is lost underneath it and also you have inadequate loading of your Stan once in a while sometimes you can save your sample or have enough in your well to actually do two electrophoresis but other times you kind of just lost some of your sample sometimes in your big hurry purifying your DNA you will leave a little bit of ethanol over your pellet and then dissolve it in your appropriate buffer usually te buffer if there's too much ethanol or isopropanol or some organic solvent when you go to actually load your gel you'll notice that your sample actually float out of the well into the buffer and so here's this little video showing that here's a quick example what happens when you don't put your tip into the well as you can see the weighting agent weighs your material down into the well but periodically you'll see sample spilling onto the surface this is usually not a huge deal but it can contaminate local wells which could lead to false readings so just be concerned of where your tip is and try to put it a little bit into the well to ensure proper loading last but not least here's a quick example what happens when you do not remove the tip before you pull the plunger back up you'll load some of your sample back into the tip this actually may not be a big deal but if you're trying to be quantitative it may lead to false results

Channel: Greg Petersen

Views: 154,884

Rating: 4.8958097 out of 5

Keywords: DNA, electrophoresis, sample, loading, Kirkwood, community, college, cedar, rapids, iowa, horizontal, agarose

Id: tTj8p05jAFM

Length: 3min 30sec (210 seconds)

Published: Wed Sep 23 2009