1. Biotechnology Lab
Bio 11
Week 1

2. 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

3. 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)

4. Prerequisites to lab Pipette tutorial (second)
Metric system review (first)

5. Metric conversions 500µL = ______mL .1mL = ________µL 10mL= ________µL

6. Micropipettors Are fragile Expensive Precise
They depend on correct usage for accuracy

7. Figure 5a: P-20 Model 6.86 m l
= or 6.86 x 10-3 ml
Figure 5b: P-200 Model m l
= or x 10-1 ml
Figure 5c: P-1000 Model 262 m l
= or 2.62 x 10-1 ml

8. 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

9. Lab Concepts in Detail

10. 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

11. Plasmids contain nonessential (but important) genes
“Bonus Package” 1: origin of replication and cloning site

12. Plasmids can be cut with restriction enzymes
Enzymes homodimerize to make symmetrical cuts
C G G A T C C A
G C C T A G G T
BamHI CG
GCCTAG
GATCCA GT
“sticky ends”

13. Restriction Enzymes cut very specific sequences of DNA

14. Plasmid DNA manipula-tion is at the heart of biotech-nology
Bacterium
Cell containing gene
of interest
Gene inserted into
plasmid
Bacterial
chromosome
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
Gene of
interest
Protein expressed
by 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

15. BamHI BamHI kanR pKAN pAMP HindIII ampR HindIII Ori Ori
Restriction digest
Ori
BamHI
Ori
ampR
BamHI
HindIII
HindIII
(2332 bp)
(3755 bp)
kanR
BamHI
HindIII
Ligation
(784 bp)
(1875 bp)
BamHI
kanR
HindIII
ampR
Ori