Biotechnology Lab

Bio 11Week 1

Brief Overview of Lab Objectives

1. Obtain Bacterial DNA (plasmids-pAMP and pKAN)2. Cut DNA into specific pieces using special enzymes

(restriction enzymes- BamHI; HindIII)3. Measure size of pieces cut by enzymes (gel

electrophoresis)4. Glue pieces together using other enzymes (DNA

ligase)5. Take glued pieces and put them into another

bacterium (plasmid transformation of E. coli)6. Separate bacteria with plasmid from those without

Today’s Objectives

1. Obtain Bacterial DNA (plasmids-pAMP and pKAN)

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

3. Extract eukaryotic genomic DNA from strawberries*

* Optional (Time permitting)

Prerequisites to lab

• Pipette tutorial (second)• Metric system review (first)

Metric conversions

• 500µL = ______mL• .1mL = ________µL• 10mL= ________µL• 10.0µL= ________mL• 1000µL= ________L

Micropipettors

Are fragile Expensive PreciseThey depend on correct usage for accuracy

Figure 5a: P-20 Model 6.86 m l

= 0.00686 or 6.86 x 10-3 ml

Figure 5b: P-200 Model 132.4 m l

= 0.1324 or 1.324 x 10-1 ml

Figure 5c: P-1000 Model 262 m l

= 0.262 or 2.62 x 10-1 ml

Steps in proper pipette usage1. Adjust volume2. Select tip3. Depress plunger to first stop4. Put tip into desired liquid5. Release plunger slowly6. Put tip into desired container7. Depress plunger to second stop8. Remove tip from container9. Discard empty used tip10.Repeat

Lab Concepts in Detail

Two Types of DNA in E. coliChromosomal 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

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

Plasmids can be cut with restriction enzymesEnzymes homodimerize to make symmetrical cuts

CGGCCTAG

GATCCAGT

“sticky ends”

C G G A T C C AG C C T A G G T

BamHI

Restriction Enzymes cut very specific sequences of DNA

Plasmid DNA

manipula-tion is at the heart

of biotech-nology

Bacterium

Bacterialchromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

pAMP pKAN

ampR

BamHI

HindIII

OriHindIII

BamHI

Ori

kanR

Restriction digestBamHI

HindIII

BamHI

HindIII

OriOri

BamHIHindIII

ampR

kanR

ampR

kanRBamHI

HindIII

Ori

Ligation(784 bp)

(3755 bp)(2332 bp)

(1875 bp)