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Session 10: Restriction Endonuclease Digestion of DNA

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by

Casey Sutton

on 7 December 2011

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Transcript of Session 10: Restriction Endonuclease Digestion of DNA

Session 10:
Restriction Endonuclease Digestion of DNA Procedure (Continued) Purpose Procedure By Casey Sutton and Kayla Vinje Bibliography Work with restriction enzymes to create rections
Prepare three samples of DNA for reactions:
1. pBR322 cut with EcoR1
2. pUC19 cut with EcoR1
3. lambda phage cut with HindIII
1. Set up reactions according to table:






2. Place each in seperate centrifuge tubes and label each tube as appropriate.
3. If did not have values in table, calculate as:
-Volume needed = (mass desired) / (concentration on vial)
-Volume needed = 200 ng / (concentration in ng/ul)
pBR322 & pUC19 restriction enzymes
cut DNA
recognize short specific recognition sequences of DNA Issues with restriction enzymes 1. Expensive
2. Relatively unstable
3. Specific
4. Need to be used correctly 4. Keep all DNAs and other reagents on ice
5. When done preparing reactions place tubes in styrofoam boat to float in waterbath at 37 degrees centigrade for 45-60 minutes
6. After incubation, add 5 ul of loading dye containing EDTA which ties up (chelates) magnesium ions, halting the activity of DNases. ... small DNA Methylation-Based
Forensic Tissue Identification Biological material is found on crime scenes

Detect tissues by methylation patterns: use fluorescent labeled primers

The DNA samples are subjected to digestion via restriction endonuclease

Restriction endonuclease will cut the tissue and add a primer

Put tissue through electrophoresis and detect methylation pattern

Allows combining tissue identification with profiling in a single procedure Austerberry, C., et al. "General Biology: Molecular and Cellular Laboratory." Creighton University (2011): 87-92. Print.
Frumkin, D., et al. “DNA methylation-based forensic tissue identification.” Forensic Science International: Genetics 5 (2011): 517-524. Print.
(http://www.youtube.com/watch?v=yc-s-WojU5Y) Pipetting Use the P20 Pipetman
Water and buffer must be added first in order for the enzyme to have an appropriate environment
Mix contents of each tube after enzyme is added by swirling pipette tip Calculating Volumes for Reactions If did not have values in table, calculate as:
-Volume needed = (mass desired) / (concentration on vial)
-Volume needed = 200 ng / (concentration in ng/ul)
Three tubes of DNA labelled pBR322, pUC19, and lambda
Each tube has concentration on label of tube
Need a final volume of 100 ng for each reaction
Full transcript