Genomic C-terminal Tagging by PCR-based Heterologous
Modules in S pombe
Step 1 PCR amplification of fragment for transformation
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Primers: for C-terminal Myc or GFP tagging, each primer's length is 100bp,
5' primer contains 80bp which corresponds to the sequence immediately upstream
of the stop codon of the target gene, 3' primer's location leaves about
200 bp space from stop codon of the target gene. Both primers contain
20 bp at their 3' end which are universal sequences for amplification of
the heterologous modules. Primers are synthesized and PAGE purified by
Integrated DNA Technologies.
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PCR reaction mixture (50 ul): We use Advantage 2 system from Clotech for
High fidelity amplification.Plasmid pFA6a-13Myc-kanMX6 (C306/1) and pFA6a-GFP(S65T)-kanMX6
(C222/4) as PCR templates
10 x reaction buffer
5 ul
4x dNTP (2.5mM)
5ul
5' primer (10 uM)
5ul
3' primer (10 uM)
5ul
Plasmid DNA ( mini prep)
0.5 ul (~100ng)
50x Ad 2 polymerase
1 ul
dd H2O
28.5 ul
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tolal volume
50 ul
Initial denature 2min 94
5 cycles of following
95 1min
50
2min
68
3min
then 20 cycles of following
95
1min
68
3min
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Pool 5 tube of each PCR reaction which contain more than 20 ug of DNA product
and subject to phenol:chloroform:isoamylalcohol extraction, precipitate
with 0.3M sodium acetate and ethanol. Wash pellet with 70% ethanol and
dissolve in 20 ul H2O.
Step 2 Transformation of S. pombe strain TEY495 (Leu-,Ura-, Ade- , DS448/2)
and Selection of G418-resistant
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Inoculate fresh DS448/2 cells into 15 ml YES medium, culture overnigt at
30 degree, monitor the cell density by heamocytometer, stop culture once
cell density come to ~1x 107/ml.
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Spin down the culture which contains ~1x 108 cells, wash with sterile water
once, transfer cells to 1.5 ml eppendorf tubes, wash with 1ml 1xLiAc/TE
once, spin down cell, get rid of supernatant.
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Add 10 ul carrier DNA and 10 ul PCR products to the cell slurry, mix well
with pipette.
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Incubate ar RT for 10min
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Add 260 ul 40% PEG/LiTE, resuspend cell gently, incubate for 1 h at 30
degree.
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Add 43 ul DMSO, cells are heat shocked for 5 min at 42 degree
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wash cell with H2O, resuspend in 0.5 ml H2O, spread 250ul/ each transformation
on YES plates
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Incubate at 30 for ~18h
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Replicate cells to 25 ug/ml G418 selection plates
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Incubate 2~3 days at 30 degree
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Pick large colonies and re-streak on fresh selection plate.
Step 3 PCR screening resistant colonies and Western blot confirmation
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For PCR, one primer locates in the target gene out of the 5' targeting
sequence, another in the Kan+ resistent gene. We use yeast colony PCR for
tagging Screening
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add approximately one forth of a 2mm colony to a 50 ul PCR reaction. Denature
for 10 min at 94 C, then cycle according to the primer melting temperature.
35 cycles. Load 10 ul on gel.
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For Western blot, we use 9E10 for Myc tagging and anti GFP for GFP tagging.
method same as common immunoblot.