New Knockout Cassettes by Brandon Davies
(see Yeast 15, 1541-1553 (1999))
The New drug resistance cassettes we got from EUROSCARF are as follows (Yes, we
got some others, but someone else can write a protocol for them):
pAG25 (natMX4) aka pJR2522
pAG35 (natMX3) aka pJR2525
pAG29 (patMX4) aka pJR2523
pAG31 (patMX3) aka pJR2526
pAG32 (hphMX4) aka pJR2524
pAG34 (hphMX3) aka pJR2527
These plasmids were built using the same pFA6 backbone as the common KanMX
plasmids and should work the same (but there seems to be a glitch, see Primer Design
below).
Resistances are as follows
 natMX confers resistance to clonNAT (use 100 μg/ml)
 patMX confers resistance to bialaphos or glufosinate and requires some funky
plates so go read the paper. 600 µg/mL in 1.7 g/L YNB w/o (NH4)2SO4 w/o
amino acids, 1 g/L L-Proline, and 20 g/L Dextrose.
 hphMX confers resistance to hygromycin B (use 300 μg/ml)
Primer Design:
Although, in theory, you should be able to use the same primers you would use
with the KanMX cassettes, I found that the recommended primer sequence for the second
primer had 4 bases that were not homologous to the sequence of the new cassettes. So I
redesigned the primer sequence so it would be compatible with the new plasmids as well
as the classic KanMX cassettes. The first primer sequence remains the same.
 For pFA6 plasmids
o 5’-(40 bp gene-specific sequence) CGGATCCCCGGGTTAATTAA-3’
o 5’-(40 bp gene-specific sequence) CGATGAATTCGAGCTCGTTT-3’
Primers for Colony PCR:
I designed the following primers to check specifically for integration of the hphMX or
natMX cassettes using colony PCR. (Naturally, you have to provide the matching gene
specific primer. I can’t do everything for you). If you are nice, I could let you borrow
them. The KanB primer will work for all the cassettes.
hphMX (binds 512 bp (hphMX4) or 958 bp (hphMX3) from upstream primer)
5’- CCCTCCGAGAGCTGCATCAGGTCGG-3’
natMX (Binds 500 bp (natMX4) or 970 bp (natMX3) from upstream primer)
5’-CCCATCCAGTGCCTCGATGGCC-3’
For more info go to http://www.uni-frankfurt.de/fb15/mikro/euroscarf/data/Del_plas.html