RESTRICTION DIGESTION AND ANALYSIS OF LAMBDA DNA.
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Transcript of RESTRICTION DIGESTION AND ANALYSIS OF LAMBDA DNA.
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RESTRICTION DIGESTION AND ANALYSIS OF LAMBDA
DNA
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SAFETY FIRST
• WEAR GLOVES• WASH HANDS WITH SOAP WHEN DONE• HAPPY BIRTHDAY
JUMP TO SLIDE 22
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What was the point of this lab?
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What is lambda(λ)? Why lambda?
• Lambda is a bacteriophage, virus that infects bacteria• Inserts its nucleic acid into the host bacterial cell • Replicates rapidly inside host cells
until the cells burst and release more phages
• Harmless to man and other eukaryotic organismsSOOOO, excellent source of DNA for experimental study.
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Lambda genome is about 48,000 bp
If linear lambda DNA is cut with HindIII, how many fragments will there be? Longest piece? Shortest piece?
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What is a restriction enzyme?
• Enzymes that cut DNA at specific places known as restriction sites
• Also called endonucleases• Bacteria use them as a natural defense against
bacteriophages• Biotechnology – cutting genes from one
organism and pasting them into another – would not be possible without these enzymes
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Enzyme Site Recognition
• Each enzyme digests (cuts) DNA at a specific sequence = restriction site
• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)
Palindrone
Restriction site
Fragment 1 Fragment 2
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5 vs 3 Prime Overhang
• Generates 5 prime overhang• DNA from any
organism cut with the same enzyme will produce complementary sticky ends
• When mixed together, complementary bases will hydrogen bond
• Ligase is needed to reform the phosphodiester bonds.
Enzyme cuts
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Common Restriction Enzymes
• EcoRI– Escherichia coli– 5’ overhang
• HindIII– Haemophilus influensae– 5’ overhang
• PstI– Providencia stuartii– 3’ overhang
5’ CTGCAG 3’
3’ CACGTC 5’
5’ GAATTC 3’
3’ CTTAAG 5’
5’ AAGCTT 3’
3’ TTCGAA 5’
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How is a restriction digest done?
• Restriction Buffer provides optimal conditions for enzyme
• Why incubate at 37°C?• Body temperature is optimal for these and most
other enzymes• What happens if the temperature is too hot or cool? Too hot = enzyme may be denaturedToo cool = enzyme activity lowered, requiring longer digestion time
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How can we separate all those fragments of DNA?
• Agarose gel electrophoresis• Agarose is purified agar–Derived from seaweed– agar provides a medium on which bacteria
(and other microorganisms can grow)– agarose provides a “sieve” for separating
DNA fragments by size• Large fragments travel slower than small
fragments
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Gel electrophoresis– DNA is negatively charged– when it’s in an electrical field it
moves toward the positive side
+–
DNA
“swimming through Jello”
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AgaroseElectrophoresis
Loading
• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode
Power Supply
Buffer
Dyes
Agarose gel
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How can we see the DNA fragments – DNA is not colored?
• Stain them!• The stain we use is relatively nontoxic, easy
to handle and dispose of• BUT it’s not very sensitive so in “real life”
other types of stains are used • Loading dyes and tracking dyes do not stain
DNA; they just help you see where your sample is and how far DNA fragments have probably traveled
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How can we determine the sizes of the DNA fragments?
• Run a standard in one of the wells; also called marker or ladder
• Standard has been cut with restriction enzymes and the size in base pairs (bp) has been determined
• Compare migration of fragments whose size is known to the migration of fragments whose size is unknown
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How can we determine the sizes of the DNA fragments?
• Create a standard curve using the migration of the marker DNA fragments
• Determine the size of the unknown fragments from this graph
• Semi-log graph paper will be needed
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Analysis of Stained Gel
Determinerestriction fragmentsizes
• Create standard curve using DNA marker
• Measure distance traveled by restriction fragments
• Determine size of DNA fragments
• Identify the related samples
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Molecular Weight Determination
Size (bp) Distance (mm)23,000* 11.0 9,400 13.0
6,500 15.0
4,400 18.0
2,300 23.0
2,000 24.0 100
1,000
10,000
100,000
0 5 10 15 20 25 30
Distance, mm
Siz
e, b
ase
pai
rsB
A
Fingerprinting Standard Curve: Semi-log
*This fragment falls outside the linear portion of the curve. You may choose to exclude it from your best fit line
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OVERVIEW• Page 21 You can answer (3 questions)• Page 22 You can answer (4 questions)• Page 23 You can answer (2 questions)
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LESSON 1• Page 24 Let’s answer the two questions in the
middle of the page• Be sure to fill out the chart• Page 25 You can answer – 2 questions at the
top and the 4 review questions at the bottom• Page 26 You can answer – 4 questions• Page 27 You can answer – 4 questions
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LESSON 2• Page 30 You can answer – 5 questions• Page 31 You can answer – 2 questions• Page 32 At the top – Let’s answer that now
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LESSON 3 • follow procedure 2. a on page 35• Page 36 Do not attach your tracing here; hand in separately;
if you have no data another group will share with you – be sure to document where the data came from
• Page 37; if you followed directions the wells are in this order:Lane 1 Marker (we know the size of these fragments; they were cut with HindIII )Lane 2 Uncut lambdaLane 3 lambda cut with PstILane 4 lambda cut with EcoRILane 5 lambda cut with HindIII
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LESSON 3
• Measure from the front of the well to the front of the band
• RECORD DATA ON PAGE 38
• BE CAREFUL GEL IS FRAGILE!
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LESSON 3 • Page 39 You do – 6 questions• Page 40 You do – 2 questions• Page 41 You do – 1 question• Clarification step 3 use marker• Page 42 – Complete the graph – you may ignore the
23,000 bp piece when drawing the best fit line• Page 43 in the “estimated” columns also include the data
from the first gel analysis in parenthesis; the number from the standard curve graph should not be in parenthesis
• Page 44 You do – 4 questions
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• Lane 1: marker, lambda cut with HindIII
• Lane 2: uncut lambda• Lane 3: lambda cut
with PstI• Lane 4: lambda cut
with EcoRI• Lane 5: lambda cut
with HindIII
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• Lane 1: marker, lambda cut with HindIII; 7 sites, 8 pieces
• Lane 2: uncut lambda; 48,502 bp
• Lane 3: lambda cut with PstI; 28 sites, 29 fragments
• Lane 4: lambda cut with EcoRI; 5 sites, 6 fragments
• Lane 5: lambda cut with HindIII
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• WHY DIDN’T WE SEE ALL THE FRAGMENTS AS BANDS IN THE GEL?
• SOME BANDS ARE SO CLOSE IN SIZE THEY DID NOT SEPARATE USING THIS PROTOCOL
• SOME FRAGMENTS ARE SO SMALL THEY CAN NOT BE DETECTED
• How could we get better results?• Change gel concentration• Longer run time• More sensitive DNA stain
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• EXPERIMENTAL ERROR• *Incorrect measurement• *Not using optimal temperature
for enzyme for the right amount of time
• *Some of sample did not enter well
• *Stock solutions not kept on ice