5/08/15

Design primers

Gene PHO85

5’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast.
5’-TATCATTATATATACATGGCTACGGTTTTTCGCTGACGGGCTGCGATAATCATTTGCA
TCCATACATTTTGATGGC -3’

3’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast.
3’-AAGGGATATATAGCGCGGCAAACTGGGCAAACTTGAGCAATACCACAGCAGTATAG
CGACCAGCATTC-5’

- tail homology PHO85
- Primer Kanamycin

Gene PHO80

5’Primer of Kanamycin resistance gene with tails using to transformation with the PHO80 gene of the yeast.
5’-ATCATAAGACGAGGATATCCTTTGGAGACTCATAGAAATCATAATCATTTGCATCCAT
ACATTTTGATGGC-3’

3’Primer of Kanamycin resistance gene with tails using to transformation with the PHO80 gene of the yeast.
3’-CTCAATCATGATTGCTTTCATAATACCCCACGAAAAATCACAGCAGTATAGCGACCA
GCATTC-5’

- tail homology PHO80
- Primer Kanamycin

Gene FRT + PHO85

5’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast, including FRT sequence to delete both of PHO80 and PHO85.
5’-TATCATTATATATACATGGCTACGGTTTTTCGCTGACGGGCTGCGGAAGTTCCTATTC
TCTAGAAAGTATAGGAACTTCATAATCATTTGCATCCATACATTTTGATGGC-3’

3’Primer of Kanamycin resistance gene with tails using to transformation with the PHO85 gene of the yeast, including FRT sequence to delete both of PHO80 and PHO85.
3’-AAGGGATATATAGCGCGGCAAACTGGGCAAACTTGAGCAATACCACTTCAAGGATAT
GAAAGATCTCTTATCCTTGAAGCAGCAGTATAGCGACCAGCATTC-5’

- tail homology PHO85
- FRT
- Primer Kanamycin

12/08/15

Culture

The Saccharomyces cerevisiae SK1 was thaw, and sow 100µL of it on YPD medium overnight. (at 30°C)
This yeast will be transformed.

PCR

3 PCR were realized on HO-Poly-KanMX4-HO plasmid to create a Kanamycin resistance marker, thanks to 3 pairs of primers wich have tails we’ll be use to knock out genes PHO80, PHO85 and both in the yeast.

Protocol:

	PHO80	PHO85	FRT+ PHO85
Master mix (µL)	50	50	50
H2O DNAse Free (µL)	45	45	45
Resistance plasmid (µL)	1	1	1
PHO80 5'Primer (µL)		2
PHO80 3'Primer (µL)		2
PHO85 5'Primer (µL)	2
PHO85 3'Primer (µL)	2
PHO85 + FRT 5'Primer (µL)			2
PHO85 + FRT 3'Primer (µL)			2

13/08/15

PCR purification

Protocol:

Dilute PCR product (5 or 10 times ?) with the resuspension buffer

Pour it in a purification column

Centrifuge 30sec at 14K rpm

Throw the filtrat

Add 700µL of EtOH (washing solution)

Centrifuge 30sec at 14K rpm

Throw the filtrat

Add 500µLof washing solution

Centrifuge 30sec at 14K rpm

Throw the filtrat

Centrifuge 30sec at 14K rpm

Throw the filtrat

Put the column in a Eppendorf

Add 45µL of RNAse/DNAse free water right on the membrane

Wait 2min

Centrifuge 2min at 10K rpm

PCR The PCR control with an electrophoresis

We expected bands around 1.300bp. The band corresponding to marker with FRT is bigger than the two others strips wich have just the Kanamycin resistance with tails.

pre-culture

Swo one colony Saccharomyces cerevisiae SK1 of the yesterday in 5mL of liquid YPD medium, let 's grow overnight.

14/08/15