1. Biotech Tools Review 1

2. Biotech Tools Review Recombinant DNA / Cloning gene DNA library
restriction enzyme, plasmids, bacteria
DNA library
cDNA library
DNA hybridization
Gel electrophoresis
RFLP
Southern blotting
PCR
Sanger sequencing
Microarray

3. You want to produce human insulin cheaply for diabetics
Find your gene
collect mRNA from pancreas cells
What to do with the mRNA? 3

4. You want to produce human insulin cheaply for diabetics
What to do with the mRNA?
make DNA
mRNA  cDNA
reverse transcriptase
insert cDNA into bacteria using plasmids
restriction enzyme
ligase
transformation
How do you find which bacteria carry the recombinant plasmids? 4

5. You want to produce human insulin cheaply for diabetics
How do you find which bacteria carry the recombinant plasmid?
amp resistance gene on plasmid
LacZ gene plasmid
plasmid
amp
resistance
LacZ gene
EcoRI
BamHI
HindIII
Which colonies do we want? White ones!! 5

6. You want to produce human insulin cheaply for diabetics
How do you find which bacteria carry the insulin gene on the plasmid?
make an insulin gene probe
tagged probe
hybridization 5’ 3’
labeled probe
genomic DNA G A T C 6

7. What if you want to find out the full sequence of the human insulin gene?
Sanger sequencing
dideoxynucleotides 7

8. What if you want to make a lot of copies of your sequenced gene?
PCR
thermocycler
primers
Taq polymerase 8

9. You want to see if 2 people have differences in their insulin genes?
Gel electrophoresis & RFLP
restriction digest
run samples on gel
see if there is a polymorphism 9

10. What if I want to locate a sequence on an electrophoresis gel?
Southern blot
tagged probe 10

11. You want to test effect of drug on gene expression in pancreas?
Microarray
collect mRNA
mRNA  cDNA
hybridize on GeneChip 11

12. Any Questions?? 12