restriction digest laboratory restriction fragment length polymorphism
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Restriction Digest Laboratory
Restriction fragment length polymorphism
ReminderYou 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 for RESULTS that will be part of your in-lab report interpretation.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 reportThe steps in BLUE you will complete outside of class as part of your data analysis.
What is:A restriction enzyme(s)?
An endonucleaseWe will focus on type II.
A restriction digest?
Restriction Enzyme Digest
Examples of Restriction Enzymes
http://www.accessexcellence.org/AE/AEC/CC/re_chart.php http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp Links to restriction enzymes:http://www.neb.com/nebecomm/EnzymeFinder.asp?
Gel Electrophoresis Following Digest
Analysis of DataAllows you to identify sizes of plasmid
By comparing migration of digested plasmid
To KNOWN SIZES of DNA.
Example of known sizes of DNA DNA Ladder or Markers
A map gives the size of fragmentsA map gives the number and position of cutting sitesJUST AN EXAMPLENot your map!Plasmid map1500 800606001400
Remember Plasmid is CircularCircular DNA: the number of fragments=number (N) of cutting sites
Linear DNA: number of fragments=N+1
2 cutting sites2 fragments2 cutting sites3 fragmentsPlasmid DNALinear DNA
Todays experimentRestriction of Digest of plasmid DNAusing two restriction enzymes.
Please refer to page 10 of the handout(6 groups)Each Group set up a rack with:
Reaction bufferwaterPlasmid DNANotI for AM labSfiI for AM lab
AluI for PM labHphI for PM labLoading Dye
Standard (marker or ladder) DNA
Label four microfuge tubes 14Must keep on ice
Pipette the samples as shown on page in handoutnot lab manual.
After you are finished pipetting your samplesPlace samples at 37C for 1 hour
After 1 hour you will be ready to load your gel
Restriction DigestAFTER 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
Gel ElectrophoresisLoad 25 ul per well
Run gel at 75 volts until the dye front is approximately half-way down gel.