Selected article for: "µg ml and PCR product"
Author: Václav Vopálenský; Michal Sýkora; Tomáš Mašek; Martin Pospíšek
Title: Messenger RNAs transcribed from yeast linear cytoplasmic plasmids possess unconventional 5’ and 3’ UTRs and suggest a novel mechanism of translation
  • Document date: 2018_5_17
    • ID: foskvkwn_133
    Snippet: The PBP1, PAB1 and LSM1 genes were deleted from the chromosomes of the K. lactis IFO1267 strain using a loxP-G418-loxP cassette (Guldener et al., 1996) . Briefly, the cassette for the deletion of the PBP1 gene was amplified from the pUG6 plasmid by PCR with the primers KL_pbp1-del_Rev and KL_pbp1-del_For. The PCR product was resolved by agarose electrophoresis, purified, and transformed into K. lactis IFO1267. The deletion of the PBP1 gene was ve.....
    Document: The PBP1, PAB1 and LSM1 genes were deleted from the chromosomes of the K. lactis IFO1267 strain using a loxP-G418-loxP cassette (Guldener et al., 1996) . Briefly, the cassette for the deletion of the PBP1 gene was amplified from the pUG6 plasmid by PCR with the primers KL_pbp1-del_Rev and KL_pbp1-del_For. The PCR product was resolved by agarose electrophoresis, purified, and transformed into K. lactis IFO1267. The deletion of the PBP1 gene was verified by PCR. This modified strain was transformed with the pSH65 plasmid (Gueldener et al., 2002) , incubated for 5 hours in nonselective medium and plated on solid medium supplemented with phleomycin (400 µg/ml). Monocolonies were grown on YPD plates with phleomycin for two days and subsequently tested for their ability to grow on G418 (250 µg/ml) selective plates. The excision of the G418 cassette was verified by PCR for selected colonies that did not grow in the presence of G418. These colonies were cultivated for five days with daily dilution under nonselective conditions (YPD medium only), resulting in the loss of the Cre-containing pSH65 plasmid. The resulting yeast strain, K. lactis IFO1267pbp1Δ, was used for subsequent PAB1 deletion using the primers KL_pab1-del_For and KL_pab1-del_Rev in a similar fashion with the exception of the G418 cassette excision. K. lactis IFO1267 was used for LSM1 deletion using primers KL_lsm1-del_For and KL_lsm1-del_Rev in a similar fashion with the exception of G418 cassette excision. The PCR reactions were carried out similarly (5 min at 95°C; then 30 cycles of 30 sec at 94°C, 30 sec at 55°C, and 4 min at 68°C; and . CC-BY-NC-ND 4.0 International license author/funder. It is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/325316 doi: bioRxiv preprint finally, 10 min at 68°C). The nucleotide sequences of the primers used for the verification of the gene disruption cassettes are summarized in Table S2 . Tab. S1

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