PRELAB DISCUSSION #12
ANNOUNCEMENTS
• CH6 Short Report due this week covering ONLY CH6AB.
• All work related to CH6C will be completed DURING your last lab session.
Please bring your computers since you will need to do a standard curve.
LAB GRADING:
• All lab points will be posted as points on blackboard by Tuesday 12/13
9 pm for you to check for any discrepancy with your own records.
• I will be available on Wed 12/14 for you to bring any grade related
issues to my attention. I will post the exact timing next Monday, but
will be some time between 9:30 am -2 pm.
• On Thu 12/15 the 421/527/621 teaching team will have a meeting to
go over lab points and finalize any adjustments (such as normalization
across sections, any outstanding student requests/ issues, etc.) and
final lab points (out of 700) will be posted by Fri 12/16. NO letter
grades will be assigned to total lab points.
CH6C WK 2
What will you do? (procedure)
●
●
Perform restriction digests using Ahd I and Pvu II
endonucleases
Perform agarose gel electrophoresis on digested plasmids
Why will you do it? (purpose)
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To create a restriction map for the isolated plasmids
To determine the identity of each plasmid (which one is
pGEM3-Rel (gene in reverse orientation to ORF) and which
one is pGEM4- Rel (gene in correct orientation to ORF)
YOUR PLASMIDS (A&B)
SP6
Pvu II 0.55
Promoter
SP6
Promoter
AmpR
Rel
pGEM3-Rel
5.27 Kb
Ahd I 3.57
AmpR
Pvu II 1.92
ori
Pvu II 2.50
•
•
•
•
•
pGEM4-Rel
5.27 Kb
Ahd I 3.57
ori
Each plasmid contains:
Pvu II 2.50
Rel gene
Ampicilin resistence gene
SP6 Promoter
Origin of replication
Restriction enzyme recognition sites
Rel
RESTRICTION ENDONUCLEASES (RE)
Enzymes that recognize and digest DNA by cleaving
phosphodiester bonds between nucleotides
Genomic DNA is protected from digestion in the cell by
DNA methylation
There are three types of endonucleases:
1. Type I: Cleave DNA ast sites >1000 bps from the
recognition site (require ATP)
2. Type II: Cleave DNA within a specific short sequence
of bases called palindromes (does NOT require ATP)
3. Type III: Cleave DNA ~25 bp from recognition
sequence (require ATP)
Restriction Endonucleases (RE) used in CH6C
Ahd I:
Puv II:
restriction site
restriction site
• Ahd I cuts the double strands asymmetrically, leaving
protruding ends. These protruding bases are referred to
as sticky ends.
• PuvII enzyme cuts the double strands symmetrically,
leaving blunt ends
• The recognized sequences are the same on both strands
when each strand is read 5’ -> 3’. Such symmetrical
sequences are called palindromes.
PRODUCTS OF A RESTRICTION DIGEST
Starting with a circular DNA (like your plasmids):
• If there is a single recognition site for a RE and the DNA is
incubated with the RE:
DNA cut once  linear DNA size same as circular DNA
• If there are multiple recognition sites for a RE and the DNA
is incubated with the RE:
# cuts= # restriction sites= # of fragments produced
(total size of fragments = size of circular DNA)
Multiple REs can be used to digest a single plasmid
simultaneously as long as the reaction conditions for the
digestion are compatible.
DETERMINING THE NUMBER and SIZE of
DIGESTED FRAGMENTS
• Run an agarose gel (typically 0.5-2%)
Percentage determined by the expected fragment sizes –
higher percentage for smaller fragments
• Visualize DNA bands
What’s going to determine the location of
the DNA bands on the gel?
• Size (# of bps): small pieces migrate faster than bigger pieces.
• Conformation (shape): Comparing 3 pieces of DNA that are
the same size. Supercoil < Linear < Nicked (relaxed) circular
• After digestion all fragments are linear.
• Charge: DNA is negative because of phosphate groups and
have m/e that depends only on size.
DETERMINING THE NUMBER and SIZE of
DIGESTED FRAGMENTS
• Plot migration distance in
mm (relative migration-Rm)
vs log Size (bp) of standards
• Determine the best linear fit
to the data and use this line
(or the graph) to find the size
of the fragments
• Find the total size of the
plasmids by adding up
fragment sizes and confirm
that it has the expected
circular plasmid size.
CREATING RESTRICTION MAPS
• A restriction map is a physical map of a piece of DNA
showing recognition sites of specific restriction enzymes
separated by lengths marked in numbers of bases
• The pattern of DNA bands is characteristic for a specific
DNA sample and the restriction enzymes used to cleave it.
A banding pattern can be referred to as a DNA fingerprint.
because it is unique to that particular DNA (and the
combination of restriction fragments).
https://www.youtube.com/watch?v=8FqMUF96cPE
CH6C Workflow
• PRELAB: Calculate the amount in μl) of DNA you need for each digest
based on the DNA concentration you have determined in CH6B (You
need ~ 0.5 μg)
• Double check volume calculations from your DNA stock for each
restriction digest set-up (total reaction volume 10 μl) Record this info in
your notebook
• Prepare restriction digest reactions and incubate @ 37C for 1 hr.
• Cast a 1% agarose gel while the digest is going using a 10 well comb
• Prepare gel samples (Total 12 μl:10 μl digest + 2 μl 6X sample buffer) and
electrophoresis chamber
• Load samples (6 digests and 1 Linear DNA Minnesota Markers) and run
gel (be careful about the running direction- DNA is (-))
• Stain in EtBr for 15 min, destain in water 2 min, and image gel on UV gel
doc (take a picture and include it in your worksheet.
SAVE PLASMID for BI/CH422 CH8
RFLP and DNA Fingerprinting
• RFLP (Restriction Fragment Length Polymorphism) analysis
• RFLP+PCR for DNA Fingerprinting
• Applications
– Disease detection/prevalence
– Forensics
– Paternity
Sickle-Cell RFLP
12
DNA fingerprinting in a murder case
PCR amplify small amounts of DNA from crime scene
Digest DNA and compare pattern of bands – DNA fingerprint
DETERMINING PATERNITY
Which one is the father? F1 or F2?
DISCUSSION WORKSHEET
Please work in pairs
Example of a Restriction Digest
Below is an agarose gel electrophoresis results of a set of restriction digest
experiments. Use the information to draw a restriction map of the DNA
Is this DNA circular or linear, how do you know?
Why are there three bands in the DNA only lane (Lane 4)?
Predicting Fragmentation Pattern for pGEM3/4
Using the restriction maps provided in the manual for the two plasmids you isolated,
fill out the following two tables that summarize the number of fragments and the size
of fragments each digestion will yield for each plasmid. Use this information to sketch
out a predicted pattern of bands you would see on an agarose gel loaded with digested
samples. Note that SP6 promoter site is used as the reference (start) point for
digestions and the number near the RE site name is the distance of that site from the
reference point in kbps.
pGEM3
Digestion with
# of fragments
Fragment Sizes (bps)
# of fragments
Fragment Sizes (bps)
AhdI
PuvII
Ahd I, Puv II
pGEM4
Digestion with
AhdI
PuvII
Ahd I, Puv II
&