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(PCR)
BCH/BIOL 406
Lecture 7
Midterm
Pick up test in class
R I C D A B E
LAC Z POLYLINKER
LAC Z
PHAGE F1
pGEM-3ZfP
3199 bp
Ampicillin Resistence
Recap of Previous lab
R I C D A B E
LAC Z POLYLINKER
LAC Z
• plasmid miniprep (Qiagen Kit)
PHAGE F1
pGEM-3ZfP
3199 bp
• DNA concentration (Genomics Center)
Ampicillin Resistence
T7 promoter 6xHis
lac operator HindIII (394)
ApaLI (755)
lacI Amp(R)
Pst I (1185)
pET101/D-TOPO
5753 bp
pBR322 origin
ApaLI (2001)
Thursday
Quiz: Study material from today’s lecture
Run agarose gel
Purify PCR fragment using Qiagen PCR kit
Aliquot 10ul of purified fragment for quantitation
and sequencing at the Genomics Center.
Impact of PCR
“ PCR has transformed molecular biology
through vastly extending the capacity to
identify, manipulate and reproduce
DNA.”
Components include:
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides –dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
How does PCR work?
One PCR Cycle:
How does PCR work?
One PCR cycle: What the products
really looks like…
Template Strand
4 DNA strands
Template Strand
8 DNA strands
How does PCR work?
Three cycles…
16 DNA strands
Notice the production of double stranded, shortened PCR products (target sequence) that spans
the two primers. Our target sequences will contain the LUX AB genes.
How does PCR work?
Four cycles…
32 DNA strands
The number of DNA strands doubles after each cycle. Target sequence predominates.
How does PCR work?
After 30
cycles…
http://biology.clc.uc.edu/fankhauser/Labs/Genetics/PCR/PCR_Protocol.htm
Components for PCR
Heat-stable DNA polymerase (Taq or
Pfu polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides –dATP, dTTP, dCTP, dGTP
DNA template
Mg++, buffer components, and water
Heat-stable DNA polymerase
Taq DNA polymerase
was isolated from the
bacterium Thermus
aquaticus.
temperatures (~95oC)
used for denaturing
DNA.
Limitations of Taq Polymerase
Error rate for Taq= 1/5000 nucleotides
http://www.bio.davidson.edu/Courses/Molbio/MolStudents/spring2002/Robinson/Isocitrate-main-page.html
Lux AB Primers
3’ TACTTCAAACCTTTATAAAC 5’
5’ CACCATGAAGTTTGGAAATATTTG 3’
(Forward Primer)
(Reverse Primer)
3’ TTTTAGCTTTACTTAAATGG 5’
5’ AAAATCGAAATGAATTTACC 3’
TC
C AGAAGGTGACCAAGTTCAT-3’
I I I I I I I
C TCTTCCA-5’
CA
Primer-Dimers
Check Your Knowledge
3’ GCATTGCTACAT 5’
(Only 12 nucleotides long. Should be at least 18 nucleotides in
length)
3’ GCCGGAGTCTGGCGCGCGCGC ‘5
(Too G-C rich. Will have a high Tm value.)
3’ GGGGATTCTACCCCACGATATAGCA-5’
(Hairpin formation between GGGG and CCCC. Also, you want to
avoid 4 or more G’s or C’s in a row.)
Components
Heat-stable DNA polymerase (Taq polymerase)
Two Primers (DNA oligonucleotides)
Deoxynucleotides –dATP, dTTP, dCTP,
dGTP
DNA template
Mg++, buffer components, and water
Deoxynucleic Acids
dATP, dTTP, dGTP and dCTP should be
present in equal amounts.