restriction digest laboratory
DESCRIPTION
Restriction Digest Laboratory. Reminder. You have transformed bacteria with plasmid DNA You have isolated plasmid DNA Today you will perform an RFLP analysis & Confirm your Plasmid Isolation. This is the third and final section of your lab report. Digest plasmid DNA - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/1.jpg)
Restriction Digest Laboratory
![Page 2: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/2.jpg)
Reminder
• You have transformed bacteria with plasmid DNA
• You have isolated plasmid DNA
• Today you will perform an RFLP analysis
• & Confirm your Plasmid Isolation
![Page 3: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/3.jpg)
This is the third and final section of your lab report.
• Digest plasmid DNA
• Determine number of cutting sites
• Determine location of cutting sites
• Determine size of fragments
• Present the “map” of the plasmid in your report
The steps in BLUE you will complete outside of class as part of your data analysis.
![Page 4: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/4.jpg)
Restriction Enzyme Digest
![Page 5: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/5.jpg)
DNA Separation following Digest
![Page 6: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/6.jpg)
Markers: Size Identification
![Page 7: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/7.jpg)
RFLP provides a map of your plasmid
• A map gives the number and position of cutting sites
• A maps gives the size of fragments
![Page 8: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/8.jpg)
Remember Plasmid is Circular
• Circular DNA: the number of fragments=number (N) of cutting sites
• versus
• Linear DNA: number of fragments=N+1
![Page 9: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/9.jpg)
2 cutting sites2 fragments
2 cutting sites3 fragments
Plasmid DNA Linear DNA
![Page 10: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/10.jpg)
You must carefully follow page 3-65
• 6 groups for today’s experiment
• Each group should set up a rack with the tubes necessary for the restriction digest
• Assign a member of your group to pick up sample tubes.
![Page 11: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/11.jpg)
Obtain a rack and:
●1. Obtain new microfuge tubes and label 2-8
![Page 12: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/12.jpg)
2. Place these tubes also on your rack
Tube L= Ladder “known sizes of DNA” Tube P=Plasmid DNA “cocktail”
Tube A: AfaI Tube B: Mae I Tube C: Xma I Tube D: Loading Dye Tube W: Water
note these enzymes are different than your lab manual
![Page 13: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/13.jpg)
Place tubes …
• On ICE
![Page 14: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/14.jpg)
Pipette the samples as shown on page 3-65
![Page 15: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/15.jpg)
After you are finished pipetting your samples
• Place samples at 37C for 1 hour
• After 1 hour you will be ready to load your gel
![Page 16: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/16.jpg)
Restriction Digest
• AFTER 1 hour DIGESTION: You must add 5 ul 10X loading dye to your samples (not to the ladder (L)).
• Pre-heat all samples including ladder for 3-5 min. at 65C
![Page 17: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/17.jpg)
![Page 18: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/18.jpg)
Gel Electrophoresis
• Load 25 ul per well
• Run gel at 75 volts for 45 minutes
• Take photograph
![Page 19: Restriction Digest Laboratory](https://reader036.vdocuments.net/reader036/viewer/2022062519/568150a9550346895dbeba17/html5/thumbnails/19.jpg)