METHODS SESSION 1

• Plasmids in the yeasts

• Library construction
• DNA sequencing
• Dominant or recessive?
• Cloning of the mutant gene
• Replica plating to different media
 Selection for plasmids:

ura3- on the chromosome: needs uracil

(auxotrophic for uracil)
URA3+ on the plasmid: makes uracil
(prototrophic for uracil)
Origin of replication

In fission yeast the most common marker are ura4, ade6, and leu1
Select for plasmid loss:

5-FOA is toxic to URA3+ cells

Plasmid loss renders the cells ura3-
and 5-FOA resistant

Replica plate the cells to 5-FOA plates

with uracil
Dideoxy sequencing
Incorporation of a dideoxyribose nucleoside terminates elongation
Olds school sequencing gels
Genomic DNA library

106-107 different plasmids

copyDNA (cDNA) library

1) Purify mRNA

2) Add polyT-primer

3) Make cDNA med reverse transcriptase

4) Ligate the cDNAs into plasmids

 You have isolated a haploid yeast clone
with an interesting tomato-like phenotype

How would you proceed?

Is the mutation dominant or recessive?

How would you isolate the gene?

 Isolation of the mutated gene:

Positional mapping (laborious)

By complementation:

Isolation of a plasmid with complementing

(wild type) allele by transformation
with a cDNA or genomic library

Sequence the entire genome!

Consider the following cross:

h- ura4+ X h+ ura4-D18

100 random spores are plated on rich medium

How many colonies will be uracil auxotrophs?

How would you isolate those?