1. 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

2. Cloning a DNA segment from lambda bacteriophage

3. Cloning a DNA segment from lambda bacteriophage
Recombinant DNA technology
First recombinant protein used
as drug - insulin from E.coli (1982)

4. 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 base pairs apart so that the restriction fragments are >100 bp long

5. 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

6. 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

7. Cloning a DNA segment from lambda bacteriophage
DNA ligation into plasmid
Ligate small DNA fragments into plasmid DNA
Plasmid has single, unique EcoRI site

8. 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

9. 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

10. 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)

11. Cloning a DNA segment from lambda bacteriophage
DNA ligation into plasmid
Ligation
ATP
Positively
charged