PRELAB DISCUSSION #12
ANNOUNCEMENTS
KEY DATES:
 CH6C Labs: Dec2-8th
 CH6 Write-up: pdf due 9 pm the evening before your CH6C Lab and
hardcopy due at the beginning of CH6C lab. (The write up template
outlining what needs to be turned-in is posted on blackboard content
CH6 Materials CH6 Write-up Template.
 CH6 Write-up Addendum: pdf due 48 hrs after the end of your CH6C lab
(each TF will arrange separately for the delivery of the hardcopy). It
constitutes completing a three page addendum sheet based on your
CH6C lab data. The sheet is posted on blackboard content CH6
Materials CH6 Addendum Sheet. You are encouraged to print that out
and complete it DURING your lab time and turn it in (you do NOT need
to turn in a pdf if you turn-in a hardcopy during lab time). But if you
prefer, you can work on it until its due date.
 Lab Final: Dec 7th and 8th : Bring your calculator. Will focus on assessing
your understanding of the experimental techniques covered in 421 labs
throughout the semester in terms of collecting, analyzing, and
interpreting data
PART I
REFLECTING ON CH6AB WK 1
What did you do? (procedure)
Isolated two plasmids (A&B) from Ecoli containing the rel gene
inserted in two opposite orientations with respect to ORF
● Performed UV Absorbance measurements at 260 and 280 nms
on the isolated plasmids.
● Performed agarose gel electrophoresis on the isolated
plasmids.
●
Why did you do it? (purpose)
To obtain DNA plasmid containing the desired gene (CH6C,422)
● To perform restriction digest to create a restriction map of the
plasmids
● To determine the concentration of the purified DNA plasmids
●
PART II
PREPARING FOR 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)
●
●
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
Pvu II 2.50
Each plasmid contains:
•
•
•
•
•
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, farther than bigger
pieces.
• Conformation (shape): Comparing 3 pieces of DNA that are
the same size. Supercoil < Linear < Nicked (relaxed) circular
• Charge: DNA is negative because of phosphate groups and
have uniform m/e
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).
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 worksheet and your notebook
• Prepare restriction digest reactions
Record these set-up tables in your worksheet
• 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
CH6 Addendum Sheet
CH6 Addendum Sheet
CH6 Addendum Sheet
Example of a Restriction Digest
Plasmid pX (6000 bps) digested with REs A, B, and C
Based on the restriction map shown
on the right complete the table below
Digestion with
Enzyme A
Enzyme B
Enzyme C
Enzyme A+B
Enzyme A+C
Enzyme B+C
Enzyme A+B+C
# of
fragments
Fragment Sizes
(bps)
Reference point (start
and end point of digest)
Enzyme A
Enzyme A
Enzyme B
Enzyme B
Enzyme C
Enzyme A + B
Enzyme A + B
Enzyme A + C
Enzyme B + C
Enzyme A + B + C
Enzyme A + B + C
Example of a Restriction Digest
Plasmid pX (6000 bps) digested with REs A, B, and C
Based on the restriction map shown
on the right complete the table below
Digestion with
# of
fragments
Fragment Sizes (bps)
Enzyme A
3
1,400, 1,500, 3,100
Enzyme B
2
2,000, 4,000
Enzyme C
1
6,000
Enzyme A+B
5 (4 bands)
1,400, 500, 1,000(2),
2,100
Enzyme A+C
4
1,400, 1,500, 2,100,
1,000
Enzyme B+C
3
2,000, 1,100, 2,900
Enzyme A+B+C 6 (4 bands)
1,400, 500, 1,000(3),
1,100
Reference point (start
and end point of digest)
Example of a Restriction Digest
Plasmid pX (6000 bps) digested with REs A, B, and C
Digestion with
# of
fragments
Fragment Sizes (bps)
A
Enzyme A
3
1,400, 1,500, 3,100
Enzyme B
2
2,000, 4,000
Enzyme C
1
6,000
Enzyme A+B
5 (4 bands)
1,400, 500, 1,000(2), 2,100
Enzyme A+C
4
1,400, 1,500, 2,100, 1,000
Enzyme B+C
3
2,000, 1,100, 2,900
Enzyme A+B+C
6 (4 bands)
1,400, 500, 1,000(3), 1,100
B
A
+
C B
A
+
C
B
+
C
A
+
B
+
C
Undi
gest
lad
ed
DNA der
RFLP and DNA Fingerprinting
• RFLP (Restriction Fragment Length Polymorphism) analysis
• RFLP+PCR for DNA Fingerprinting
• Applications
– Disease detection/prevalence
– Forensics
– Paternity
Sickle-Cell RFLP
31
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?