DIY Molecular Cloning I:
DNA Overview, Extracting DNA,
Finding genetic sequences, Designing a cloning
strategy
C. Rouskey
Jan.2014
Intros
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...name and background, what you hope to
learn...
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Me
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GETit
Outline
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DNA Structure and Function/DNA Extraction
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Sequences on the Web/Determining your GOI
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Choosing a plasmid
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Components (OriC, Promoter, Reporters, GOI)
Designing a cloning Strategy
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Cloning System? Eukaryotic or Prokaryotic
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Cloning
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GETit Cloning Work
DNA
DNA Basics
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Deoxyribonucleic Acid
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Stores information
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Made up of nucleotides
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Adenine (p), Cytosine (py), Thymine (py), Guanine (p)
with a deoxyribose backbone; RNA T = U(racil)
These nucleotides bind in complement to each other via
hydrogen bonds (G:::C; A::T(U))
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Hydrogen bonds are fairly weak when taken on their own, but the
cumulative forces of these bonds make DNA stable
5' and 3' ends
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Two strands running in the opposite direction
Sequences are reverse complement
What kind of DNA?
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Prokaryotic or Eukaryotic
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Genomic
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Plasmid
DNA Replication
Where does it occur?
How does it occur (overview)?
Major enzyme?
What are the results?
DNA Transcription
Where does it occur?
How does it occur? Promoter, ATG
What are the results?
Start and Stop Codons
DNA Translation
Where does it occur?
How does it occur? Ribosomes/tRNA, Start/Stop
Codons
What are the
Results?
PROTEINS
Overview: Cloning with REs
What is molecular cloning?
tools used to create recombinant DNA
molecules
General steps:
Amplify (via PCR) gene with RE sequences present
Digest PCR product and plasmid/Purify
Ligate PCR product into Plasmid
Transform E. coli – look at expression or purify plasmid
Molecular Biologist Toolkit
Genetic Databases –
resource for sequences
Online analysis tools – primer design, restriction
assessment
DNA/RNA
– the genetic starting material
Plasmids
– functional holding place for gene
Primers
– directs DNA amplification, targets
specific genes
Enzymes
– used in the amplification cutting and
ligation of DNA.
Host Strains – amplify and express your GOI
Online Resources
Gene search: http://www.pubmed.com
Restriction analysis: http://tools.neb.com/NEBcutter2
Primer assessment:
http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/
Composite program: Serial Cloner
Plasmids
Autonomously reproducing circular DNA
Origin of replication determines copy number (OriC)
Careful not to create a bio-burden on your cells
Plasmids
Promoter - drives the transcription of GOI
Multiple cloning Site – Space to pop in GOI
downstream of promoter
Reporters – let you assess if GOI is being
expressed
Purification Tags – helpful in downstream
purification of proteins (useful tool for functional
protein studies
Primers
Strands of DNA that serve as the starting point for
DNA synthesis
Host Strains
E. coli is the basic cloning host for growing
plasmid and expressing prokaryotic proteins.
E. coli can be used to propagate plasmid
containing eukaryotic genes but expression is
not really useful (post-translational modifications
present in Euks)
Competent cells! E. cloni 10G (super easy), Can
make your own competent cells
After plasmid is made, pop it into E. coli
GETit Specific Cloning (example)
Quick GETit background
Bacteriophage therapy
Lytic vs. Lysogenic Phage
(for more info visit http://getitproject.org/getitproject-meetup-112313/
Cloning (GETit)
1. Find the genetic sequences of interest
http://www.pubmed.com
a. are there phage in the N. gonorrhoeae
genome?
Cloning Strategy
Find a plasmid that you can clone into:
What are you trying to accomplish?
What kind of gene is it?
What do you want to do with the genetic material?
Order plasmid from addgene (http://addgene.com)
We want a moderate copy number, promoter,
antibiotic resistance gene
GOI: phage tail protein I (187aa)
5'TCACACTGCACAGCCCCGCTGCCGACATCGAATGCGGCAACGGCGAATAC
ATCCGAATCACGTCCACGCTCGAGCGCGAATAAATGAACAGCACCGTCCCC
GCGAACAACAGCCCCCTGCAACACGCACTGGCAAAGCTGACAGAACGCGA
AACTGCCGCCGTCTCCCGCCAACTCGACCCCGCCCGATGCGACCCCGGAT
TTTTACCCTTTCACGCCTTCGCAAGAAGCATCGGCACGGAAGAGGGCTGG
GACTTTGCCGAAACCGACGAAGCCCGCCGCAACCTCATCGCAGGCTTTGC
CGAAATCCACGCCCGAAAAGGCACGCCGTACGCCATCCGCGCCCTCTTCC
CCATCTTGCGGCTGGGCGAAATCCAAATTATCGAACGCGACGGCGAGTTCA
AGTGGGACGGCTCGGTCTTGTTCGACGGCAGCCGCACATTCGGCAGGCG
CGAGGGTGACTGGGCGGAATACCGCATTGTCTTAACGCGCCCCGTCAGCA
TCCGCCAAACCGCCCGCATCCGCGCCATGTTGGCGGAAATCGCCCCCTTG
CGGTGCGAACTTACCGCGCTCGACTACCGCAACCATCCCCACCGCTGGAA
CGGCAAAATCCGCTTTAACGGCGAATACGGTTTCGGCACGACATAACGCGC
CCCCGAAAATCAACAAACAAAAGGAAGCCCCAAAATGGCAAACGCAACCGA
ACAAAACCAATTCGACCAAGCCGTCCGCC-3'
Enzymes
DNA Polymerase (used to amplify template)
Restriction endonucleases cut at specific sites
Ligase
Deciding where to clone?
1. I like BamHI and EcoRV (can I cut them
together?)
http://66.155.211.155/nebecomm/DoubleDigestCalculator.asp
BamHI
2. Analyze Gene Sequence
- Are there restriction sites internally?
http://tools.neb.com/NEBcutter2/!
EcoRV
Primer Design
1. Determine Primer sequence for PCR...
5' end...
Sequence:
5' TCACACTGCACAGCCCCGCTGCCGACATCGAATGCGGCAA
Primer:
5' -
CCGCTGCCGGATCCGAATG
3' end...
AAAACCAATTCGACCAAGCCGTCCGCC – 3'
Sequence: 5' -
3' - TTTTGGTTAAGCTGGTTCGGCAGGCGG – 5'
Primer:
3' - TTTTGGTTGATATCGTTCGGCAGGCGG – 5'
5' -
GGCGGACGGCTTGCTATAGTTGGTTTT – 3'
DNA Sequence 5' – TCACACTGCACAGCCCCGCTGCCGACATCGAATG -3'
Primer
5' -
Complement
3' - AGTGTGA....
Reverse Complement
5' – CATTCGAT....
CCGCTGCCGGATCCGAATG
Now...PCR
Restriction Digest
Set up a restriction digest reaction using BamHI
and EcoRV
Digest Plasmid and PCR product
Analyze digest on gel
Ligate and Transform!
18 hours later. . .
Downstream Applications
Sequencing (confirm protein)
Protein Expression/Analysis via PAGE
Protein Purification
Experiment
1. Find the genetic sequence
http://banana-genome.cirad.fr/musa
2. Design primers
3. Extract DNA from Musa acuminata...(Protocol)
4. PCR