Cloning a DNA segment from lambda bacteriophage
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Cloning a DNA segment from lambda bacteriophage. Recombinant DNA technology Allows study of the structure & function of a single protein coding gene in purified form

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Cloning a DNA segment from lambda bacteriophage

Recombinant DNA technology

Allows study of the structure & function of a single protein coding gene in purified form

Allows amplification and isolation of specific genes using simple procedures (usually involves help of bacterial cells and plasmids)

Trying to purify a specific gene from cellular DNA would require a magnitude of purification & lots of starting material



Cloning a DNA segment from lambda bacteriophage

Recombinant DNA technology

First recombinant protein used

as drug - insulin from E.coli (1982)


Cloning a DNA segment from lambda bacteriophage

Recombinant DNA technology

To isolate segment of DNA to be cloned usually need to cleave it out of larger piece of DNA

(or can PCR amplify)

How do scientists do this? Use RESTRICTION ENDONUCLEASES

RESTRICTION ENDONUCLEASES - protein enzymes that cleave the phosphodiester bonds that connect the nucleotide units in DNA or RNA at very SPECIFIC sites

These enzymes are mainly produced by bacteria where they degrade invading foreign DNA; they have been purified from these sources and are now available commercially (300 RE available)

Most restriction enzymes recognize a specific sequence of 4-6 nucleotides in DNA and each will cut the DNA into discrete pieces known as restriction fragments

If a segment of DNA has more than 1 restriction site for the same enzyme, those sites are usually 100-1000 base pairs apart so that the restriction fragments are >100 bp long


Cloning a DNA segment from lambda bacteriophage

Recombinant DNA technology

Cohesive ends very important in recombinant DNA procedures because it allows two DNA fragments to be linked together by complementary base pairing at their ends


Cloning a DNA segment from lambda bacteriophage

Recombinant DNA technology

Part 1 of lab

Digestion of lambda bacteriophage DNA with EcoRI

Digestion of pUC18 plasmid DNA with EcoRI

Other facts about restriction endonucleases

amount of enzyme usually given in units of activity

1U is usually amount of enzyme needed to digest 1 µg of phage  DNA in 1 hour at 37 ˚C in correct buffer

just like all enzymes, RE have optimal reaction conditions (pH, buffer-ionic strength, temperature)

EcoRI has a preference for buffers with high ionic strength (100 mM NaCl, 10 mM MgCl2, 50 mM Tris-HCl pH 8)

enzymes come from company in a concentrated form, stored at -20 ˚C in buffer with 50% glycerol

reason for glycerol storage - does not freeze

freeze-thaw BAD for protein enzymes

DO NOT CONTAMINATE ENZYME STOCK TUBES!!!!

Heat inactivate restriction enzyme (70 ˚C)

Ligate cleaved fragments to each other


Cloning a DNA segment from lambda bacteriophage

DNA ligation into plasmid

Ligate small DNA fragments into plasmid DNA

Plasmid has single, unique EcoRI site


Cloning a DNA segment from lambda bacteriophage

DNA ligation into plasmid

Ligate small DNA fragments into plasmid DNA

Lambda bacteriophage has many EcoRI sites

Restriction map of lambda DNA - EcoRI

Sizes of restriction fragments

21,226

4878

5643

7421

5804

3530

21,226

26,104

31,747

39,168

44,972

Position of EcoRI restriction sites


Cloning a DNA segment from lambda bacteriophage

DNA ligation into plasmid

Ligate small DNA fragments into plasmid DNA

 DNA was cut with EcoRI and pUC plasmid has been cut with EcoRI


Cloning a DNA segment from lambda bacteriophage

DNA ligation into plasmid

Ligation by DNA Ligase - in cell, ligase used in DNA replication, repair, recombination

T4 DNA Ligase - isolated from T4 phage infected E.coli (requires Mg2+ and ATP)


Cloning a DNA segment from lambda bacteriophage

DNA ligation into plasmid

Ligation

ATP

Positively

charged


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