Transcript Biotechnology Lab

Biotechnology Lab
Bio 11
Week 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
Precise
They depend on correct usage for accuracy
Figure 5a: P-20 Model 6.86
ml
= 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 usage
1. Adjust volume
2. Select tip
3. Depress plunger to first stop
4. Put tip into desired liquid
5. Release plunger slowly
6. Put tip into desired container
7. Depress plunger to second stop
8. Remove tip from container
9. Discard empty used tip
10.Repeat
Lab Concepts in Detail
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
“Bonus Package” 1: origin of replication and cloning site
Plasmids can be cut with restriction enzymes
Enzymes homodimerize to make symmetrical cuts
BamHI
CGGATCCA
GCCTAGGT
CG
GCCTAG
GATCCA
GT
“sticky ends”
Restriction Enzymes cut very specific
sequences of DNA
Bacterium
Gene inserted into
plasmid
Plasmid
DNA
manipulation is at
the heart
of biotechnology
Bacterial
chromosome
Cell containing gene
of interest
Plasmid
Gene of
interest
Recombinant
DNA (plasmid)
DNA of
chromosome
Plasmid put into
bacterial cell
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
Basic
research
on gene
Gene for pest
resistance inserted
into plants
Protein harvested
Basic research and
various applications
Gene used to alter
bacteria for cleaning
up toxic waste
Protein dissolves
blood clots in heart
attack therapy
Basic
research
on protein
Human growth hormone treats stunted
growth
BamHI
BamHI
pKAN
pAMP
HindIII
ampR
Ori
BamHI
kanR
ampR
Restriction digest
Ori
Ori
Ori
BamHI
(2332 bp)
HindIII
(3755 bp)
BamHI
HindIII
HindIII
(784 bp)
kanR
Ligation
(1875 bp)
BamHI
kanR
ampR
HindIII
Ori
HindIII