DIY Molecular Cloning I
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DIY Molecular Cloning I : DNA Overview, Extracting DNA, Finding genetic sequences, Designing a cloning strategy. C. Rouskey Jan.2014. Intros. ...name and background, what you hope to learn... Me GETit. Outline. DNA Structure and Function/DNA Extraction

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Diy molecular cloning i dna overview extracting dna

DIY Molecular Cloning I:

DNA Overview, Extracting DNA,

Finding genetic sequences, Designing a cloning strategy

C. Rouskey

Jan.2014


Diy molecular cloning i dna overview extracting dna

Intros

  • ...name and background, what you hope to learn...

  • Me

  • GETit


Diy molecular cloning i dna overview extracting dna

Outline

  • DNA Structure and Function/DNA Extraction

  • Sequences on the Web/Determining your GOI

  • Choosing a plasmid

    • Components (OriC, Promoter, Reporters, GOI)

  • Designing a cloning Strategy

    • Cloning System? Eukaryotic or Prokaryotic

  • Cloning

  • GETit Cloning Work


Diy molecular cloning i dna overview extracting dna

DNA


Diy molecular cloning i dna overview extracting dna

DNA Basics

  • Deoxyribonucleic Acid

    • Stores information

    • Made up of nucleotides

      • 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))

        • Hydrogen bonds are fairly weak when taken on their own, but the cumulative forces of these bonds make DNA stable

    • 5' and 3' ends

      • Two strands running in the opposite direction

      • Sequences are reverse complement


Diy molecular cloning i dna overview extracting dna

What kind of DNA?

  • Prokaryotic or Eukaryotic

    • Genomic

    • Plasmid


Diy molecular cloning i dna overview extracting dna

DNA Replication

Where does it occur?

How does it occur (overview)?

Major enzyme?

What are the results?


Diy molecular cloning i dna overview extracting dna

DNA Transcription

Where does it occur?

How does it occur? Promoter, ATG

What are the results?


Start and stop codons

Start and Stop Codons


Diy molecular cloning i dna overview extracting dna

DNA Translation

Where does it occur?

How does it occur? Ribosomes/tRNA, Start/Stop Codons

What are the

Results?

PROTEINS


Diy molecular cloning i dna overview extracting dna

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


Diy molecular cloning i dna overview extracting dna

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

Online Resources


Plasmids

Plasmids

Autonomously reproducing circular DNA

Origin of replication determines copy number (OriC)

Careful not to create a bio-burden on your cells


Plasmids1

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

Primers

Strands of DNA that serve as the starting point for DNA synthesis


Host strains

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

GETit Specific Cloning (example)


Diy molecular cloning i dna overview extracting dna

Quick GETit background

Bacteriophage therapy

Lytic vs. Lysogenic Phage

(for more info visit http://getitproject.org/getit-project-meetup-112313/


Diy molecular cloning i dna overview extracting dna

Cloning (GETit)

1. Find the genetic sequences of interest

http://www.pubmed.com

a. are there phage in the N. gonorrhoeae genome?


Diy molecular cloning i dna overview extracting dna

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'-TCACACTGCACAGCCCCGCTGCCGACATCGAATGCGGCAACGGCGAATACATCCGAATCACGTCCACGCTCGAGCGCGAATAAATGAACAGCACCGTCCCCGCGAACAACAGCCCCCTGCAACACGCACTGGCAAAGCTGACAGAACGCGAAACTGCCGCCGTCTCCCGCCAACTCGACCCCGCCCGATGCGACCCCGGATTTTTACCCTTTCACGCCTTCGCAAGAAGCATCGGCACGGAAGAGGGCTGGGACTTTGCCGAAACCGACGAAGCCCGCCGCAACCTCATCGCAGGCTTTGCCGAAATCCACGCCCGAAAAGGCACGCCGTACGCCATCCGCGCCCTCTTCCCCATCTTGCGGCTGGGCGAAATCCAAATTATCGAACGCGACGGCGAGTTCAAGTGGGACGGCTCGGTCTTGTTCGACGGCAGCCGCACATTCGGCAGGCGCGAGGGTGACTGGGCGGAATACCGCATTGTCTTAACGCGCCCCGTCAGCATCCGCCAAACCGCCCGCATCCGCGCCATGTTGGCGGAAATCGCCCCCTTGCGGTGCGAACTTACCGCGCTCGACTACCGCAACCATCCCCACCGCTGGAACGGCAAAATCCGCTTTAACGGCGAATACGGTTTCGGCACGACATAACGCGCCCCCGAAAATCAACAAACAAAAGGAAGCCCCAAAATGGCAAACGCAACCGAACAAAACCAATTCGACCAAGCCGTCCGCC-3'

GOI: phage tail protein I (187aa)


Enzymes

Enzymes

DNA Polymerase (used to amplify template)

Restriction endonucleases cut at specific sites

Ligase


Diy molecular cloning i dna overview extracting dna

Deciding where to clone?

1. I like BamHI and EcoRV (can I cut them together?)

http://66.155.211.155/nebecomm/DoubleDigestCalculator.asp

BamHIEcoRV

2. Analyze Gene Sequence

- Are there restriction sites internally?

http://tools.neb.com/NEBcutter2/!


Diy molecular cloning i dna overview extracting dna

Primer Design

1. Determine Primer sequence for PCR...

5' end...

Sequence: 5' -TCACACTGCACAGCCCCGCTGCCGACATCGAATGCGGCAA

Primer: 5' - CCGCTGCCGGATCCGAATG

3' end...

Sequence: 5' - AAAACCAATTCGACCAAGCCGTCCGCC – 3'

3' -TTTTGGTTAAGCTGGTTCGGCAGGCGG – 5'

Primer:3' -TTTTGGTTGATATCGTTCGGCAGGCGG – 5'

5' -GGCGGACGGCTTGCTATAGTTGGTTTT – 3'


Diy molecular cloning i dna overview extracting dna

DNA Sequence 5' – TCACACTGCACAGCCCCGCTGCCGACATCGAATG -3'

Primer 5' - CCGCTGCCGGATCCGAATG

Complement

3' - AGTGTGA....

Reverse Complement

5' – CATTCGAT....


Now pcr

Now...PCR


Diy molecular cloning i dna overview extracting dna

Restriction Digest

Set up a restriction digest reaction using BamHI and EcoRV

Digest Plasmid and PCR product

Analyze digest on gel


Diy molecular cloning i dna overview extracting dna

Ligate and Transform!


Diy molecular cloning i dna overview extracting dna

18 hours later. . .


Sequencing confirm protein protein expression analysis via page protein purification

Sequencing (confirm protein)

Protein Expression/Analysis via PAGE

Protein Purification

Downstream Applications


Diy molecular cloning i dna overview extracting dna

Experiment

1. Find the genetic sequence

http://banana-genome.cirad.fr/musa

2. Design primers

3. Extract DNA from Musa acuminata...(Protocol)

4. PCR


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