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CHE 554
BIOCHEMISTRY LAB
PCR:
the polymerase chain reaction.
Restriction Analysis
Professor
Testa
5’ to 3’
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melt 95°C
Cool to 55 °C
primers bind +
incubate at 65 °C +
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primers at +
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x1000 +
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PCR, the video
Also http://www.youtube.com/watch?v=x5yPkxCLads
Thermal cycler
Reagents:
These are used in
very small
quantities. Since
(in principle) a
single copy of a
gene suffices to
transform a
bacterium, this is
quantum biology.
Plasmid Maps
9
Primer design
Sequence of pBluescript
ctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaat
aggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttgg
aacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggccc
actacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaag
ggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaagga
gcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgcc
gctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgc
tattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtca
cgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccc
cctcgaggtcgacggtatcgataagcttgatatcgaattcctgcagcccgggggatccactagttctagagcgg
ccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtc
atagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgta
aagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcggga
aacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttc
cgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcgg
taatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagg
aaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg
ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgc
gctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttct
catagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc
cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc
cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtgg
tggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaa
aagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcaga
ttacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaa
aactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatg
aagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcac
ctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgg
gagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagc
aataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctatta
attgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggc
atcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatg
atcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcag
tgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtg
actggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaat
acgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaac
tctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatct
tttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgac
acggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatga
gcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgcca
c
10
Plasmid Maps
5’CGTACCGATGGTACCGGAT3’
3’GCATGGCTACCATGGAATA5’
5‘CGTACCGATGG3’ + 5’TACCGGAT3’
3‘GCATGGCTACCAT5’ 3’GGAATA5’
Plasmid Maps
http://pathmicro.med.sc.edu/
http://en.wikipedia.org/wiki/ pcr/realtime-home.htm
16
Ethidium_bromide (A nice tutorial !)
Agarose Gel Casting and
Electrophoresis
Documenting your gel
The Experiment
For the PCR reaction, we will make up our own ‘guru’ mix.
Make this in a 1.5 μL sterile eppendorf tube.
– Sterile water: 104 μL
– 10X Buffer: 65 μL
– 50 mM MgCl2: 51 μL
– 1.25 mM dNTPs: 68 μL each
– Total 492 μL
Make up four reactions, each in a 0.5 μL sterile eppendorf tube.
– put 76 μL guru mix in each of the four tubes,
– Tube A: Add 6.8 μL Primer 1 + 10 μL template + 3 μL Taq +
5.5 μL sterile water.
– Tube B: Add 6.8 μL Primer 1 + 6.8 μL Primer 2 + 10 μL
template
– Tube C: Add 6.8 μL Primer 1 + 6.8 μL Primer 2 + 3 μL Taq
+ 10 μL template.
– Tube D: Add 6.8 μL Primer 1 + 6.8 μL Primer 2 + 3 μL Taq
+ 10 μL 10xtemplate.
– 94 °C for 2 minutes
– {ramp to 52 °C over 30 sec.
– ramp to 55 °C over 30 sec.
– 55 °C for 1 minute
– ramp to 72 °C over 10 sec.
– 72 OC for 2 minutes
– ramp to 94 °C in 15 sec.
– 94 OC for 30 sec.}
– Hold at 4 OC
Day 2
– Make a 0.7% agarose gel
Follow steps 5-7 on day 2
Use TAE buffer, not TBE
Add 3 uL Ethidium Bromide to the gel solution
Do not worry about evaporation
Column Name
1 Elani
2 Michael
4 Tara
5 Jeremy
7 Jennifer
8 Zach
10 Govind
23
Safety Considerations
Observe all normal laboratory safety practices.
Ethidium bromide is a large, flat basic molecule that resembles a DNA base pair. Because of its chemical structure, it
can intercalate (or insert) into a DNA strand. Ethidium bromide is commonly used in molecular biology laboratories to
stain electrophoresis gels. The compound forms fluorescent complexes with nucleic acids and these can be viewed
under UV light.
Ethidium bromide is a known mutagen in certain animal and microorganism test systems. Although the compound
has not been thoroughly evaluated in humans, based on current toxicity data and its interaction with DNA it should be
handled with considerable caution.
Physical Data
Description: Compound may be ordered as a solid powder, tablets, or a stock solution of known concentration.
Melting Point (solid): 500o F (260o C)
Exposure Limits
Occupational exposure limits have not been established by OSHA, NIOSH, or ACGIH.
Toxicity Data
LD50 wt/kg oral LC50 4-hr inhalation
Descriptive Term
dose in rats dose in rats
Acute Animal Toxicity Extremely Toxic < 1 mg < 10 ppm
Oral; rat: LD50 = 1503 mg/kg Highly Toxic 1-50 mg 10-100 ppm
Inhalation; rat LC50 = 0.0118-0.1340 ppm Moderately Toxic 50-500 mg 100-1,000 ppm
Subcutaneous; mouse: LD50 = 110 mg/kg Slightly Toxic 0.5-5 g 1,000-10,000 ppm
Intraperitoneal; mouse: LDLo = 20 mg/kg Practically Nontoxic 5-15 g 10,000-100,000 ppm
Relatively Harmless > 15 g > 100,000 ppm
Lab Attire: Always wear long pants, closed toe shoes, and a lab coat when handling hazardous materials.
25
Chronic
• This agent intercalates DNA strands and was mutagenic in a number of test systems (yeast cells).
• The chemical, physical, and toxicological properties have not been thoroughly investigated in humans.
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