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Biotechnology Lab. Bio 11 Week 1. Brief Overview of Lab Objectives . Obtain Bacterial DNA ( plasmids - pAMP and pKAN ) Cut DNA into specific pieces using special enzymes ( restriction enzymes- BamHI ; HindIII ) Measure size of pieces cut by enzymes ( gel electrophoresis)

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Biotechnology lab

Biotechnology Lab

Bio 11

Week 1


Brief overview of lab objectives
Brief Overview of Lab Objectives

  • Obtain Bacterial DNA (plasmids-pAMP and pKAN)

  • Cut DNA into specific pieces using special enzymes (restriction enzymes- BamHI; HindIII)

  • Measure size of pieces cut by enzymes (gel electrophoresis)

  • Glue pieces together using other enzymes (DNA ligase)

  • Take glued pieces and put them into another bacterium (plasmid transformation of E. coli)

  • Separate bacteria with plasmid from those without


Today s objectives
Today’s Objectives

  • Obtain Bacterial DNA (plasmids-pAMP and pKAN)

  • Cut DNA into specific pieces using special enzymes (restriction enzymes- BamHI; HindIII)

  • Extract eukaryotic genomic DNA from strawberries*

    * Optional (Time permitting)


Prerequisites to lab
Prerequisites to lab

  • Pipette tutorial (second)

  • Metric system review (first)


Metric conversions
Metric conversions

  • 500µL = ______mL

  • .1mL = ________µL

  • 10mL= ________µL

  • 10.0µL= ________mL

  • 1000µL= ________L


Micropipettors
Micropipettors

Are fragile Expensive Precise

They depend on correct usage for accuracy


Steps in proper pipette usage
Steps in proper pipette usage

  • Adjust volume

  • Select tip

  • Depress plunger to first stop

  • Put tip into desired liquid

  • Release plunger slowly

  • Put tip into desired container

  • Depress plunger to second stop

  • Remove tip from container

  • Discard empty used tip

  • Repeat



Two types of dna in e coli
Two Types of DNA in E. coli

Chromosomal DNA – necessary for cell survival; circular, double-stranded

Plasmid DNA – extrachromosomal DNA (“bonus material”) useful for experimental manipulation; circular, double-stranded


Plasmids contain nonessential but important genes
Plasmids contain nonessential (but important) genes

“Bonus Package” 1: origin of replication and cloning site


Plasmids can be cut with restriction enzymes

C G G A T C C A

G C C T A G G T

Plasmids can be cut with restriction enzymes

Enzymes homodimerize to make symmetrical cuts

BamHI

GATCCA

GT

CG

GCCTAG

“sticky ends”



Plasmid dna manipula tion is at the heart of biotech nology

Cell containing gene

of interest

Bacterium

Gene inserted into

plasmid

Bacterial

chromosome

Plasmid

Gene of

interest

Recombinant

DNA (plasmid)

DNA of

chromosome

Plasmid put into

bacterial cell

Plasmid DNA manipula-tion is at the heart of biotech-nology

Recombinant

bacterium

Host cell grown in culture

to form a clone of cells

containing the “cloned”

gene of interest

Protein expressed

by gene of interest

Gene of

interest

Copies of gene

Protein harvested

Basic research and

various applications

Basic

research

on gene

Basic

research

on protein

Gene for pest

resistance inserted

into plants

Gene used to alter

bacteria for cleaning

up toxic waste

Protein dissolves

blood clots in heart

attack therapy

Human growth hor-

mone treats stunted

growth


BamHI

BamHI

kanR

pKAN

pAMP

HindIII

ampR

HindIII

Ori

Ori

Restriction digest

Ori

BamHI

Ori

ampR

BamHI

HindIII

(2332 bp)

HindIII

(3755 bp)

kanR

BamHI

HindIII

Ligation

(784 bp)

(1875 bp)

BamHI

kanR

HindIII

ampR

Ori


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