Biotech outlined

gooseliverΒιοτεχνολογία

22 Οκτ 2013 (πριν από 3 χρόνια και 9 μήνες)

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Biotechnology Lab

Cloning of KanR gene

Biotechnology Techniques


Polymerase Chain Reaction (PCR)


Agarose Gel Electrophoresis


Ligation


Transformation


MacConkey Plate Agarose Gel Bacteria
Growth


Restriction Enzyme Digest


Day

1

Day 2 (part 1)

Day 2 (part 2)

Day 3 (part 1)

Day 3 (part 2)

Day 4 (part 1)

Day 4 (part 2)

Day 5 (part 1)

Day 5 (part 2)

Day 1
-

PCR


Polymerase Chain Reaction (PCR)


A technique to ‘amplify’ (copy) a piece of
DNA millions of times


Used in EVERY genetics lab



DNA fingerprinting


Cloning


Genetic Testing


Gene sequencing

Steps of PCR

1.
Pipette Quantities of Reagents into
microtube


PCR reaction mixture


Buffer


Nucleotides


DNA Polymerase


KanR DNA template and DNA Primers

2.
Load microtube into Thermocycler



Thermocycler

1.
Denature


-

‘unzip’ the double stranded DNA by increasing the
temperature (90
o
C)

2.
Anneal


-

allow the DNA primers to ‘stick’ to the DNA
around gene by decreasing the temperature (55
o
C)

3.
Extend


-

allow the DNA Taq Polymerase to locate the
primer and read the code adding nucleotides (75
o
C)




REPEAT ~ 40 times!!!!


Restriction Digest and Gel
Electrophoresis


Molecular Check


What did we expect to see on a gel if the
KanR gene was successfully inserted?

Plasmid Map


Which plasmid represents the original T
-
vector plasmid?


Which plasmid represents a successful insert of KanR?


How might it be possible for there to be two KanR genes in plasmid B?

Phenotype Analysis


Selective Plating

Ampicillin Plate

Kanamycin Plate


What E.coli will grow on the ampicillin plate?


What E.coli will grow on the kanamycin plate?


What does this indicate about the genetic material within the E.coli?

Kanamycin Plate