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DNA Technology

by: Dr. Kayli Nienow

DNA Technology IST 340

Dr. Kayli Nienow
GPA 3.52

Elizabeth Murray

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Elizabeth Murray
Class Notes
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This 0 page Class Notes was uploaded by Dr. Kayli Nienow on Sunday November 1, 2015. The Class Notes belongs to IST 340 at Marshall University taught by Elizabeth Murray in Fall. Since its upload, it has received 12 views. For similar materials see /class/233258/ist-340-marshall-university in General Science at Marshall University.


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Date Created: 11/01/15
IST 340 DNA Technology Proper Enzyme Usage Enzymes such as restriction endonucleases DNA polymerases and DNA ligases are important tools in molecular biology and biochemistry To ensure that they function optimally it is important that they are handled properly Commercial companies provide them in high concentrations in glycerolbased solutions to increase their stability during storage Enzymes are generally stored at 20 C and should be kept on ice when outside of the freezer Keeping them cold minimizes their inactivation The following procedures should be followed when handling enzymes When using enzymes keep them on ice at all times This means that you should not put a tube of enzyme on a rack on your workbench while you set up the rest of your reaction Always centrifuge microtubes of enzymes brie y before opening the tube Be careful not to touch the cap or the upper rim of the tube with your fingers You do not want to introduce nucleases that could degrade the DNA in your reactions and you do not want to introduce proteases that could degrade the enzymes in the tubes Hold the tube of enzyme with your fingers positioned below the rim but above the level of the enzyme solution The heat from your fingers could rapidly warm up the enzyme solution Holding the tube in this manner also lets you see the tip enter the enzyme solution when you are pipetting Hold the tube at eye level so you can watch as you put the pipette tip just below the surface of the solution and watch as the pipette tip fills with the enzyme solution The enzyme glycerol solution is very viscous You must pipette slowly and carefully to measure accurately DON T insert the tip deeply into the enzyme solution or the glycerol enzyme solution will coat the outside of the tip and you will add more enzyme than you need to the reactions The activity of restriction enzymes can be affected by glycerol The volume should never exceed 110 of the reaction Certain restriction enzymes can exhibit star activity under certain conditions Star activity refers to an enzyme cutting sequences other than its normal recognition sequence Star activity can be caused by high glycerol concentration or low ionic strength of buffer or cutting with excess enzyme When assembling an enzyme reaction always add the enzyme last so that it is placed in the correct concentration of buffer Do not vortex enzyme mixtures Hold the tube in one hand and ick it several times with your finger Centrifuge the tube brie y to bring the droplets off the side of the tubes to the bottom


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