nmt1 full strength, Full repression: 15uM thiamine (5ug/ml). Full induction: no thiamine. Takes about 18 hours. Partial induction: 0.05uM thiamine (0.016ug/ml). For more information about titrating nmt, and the different levels of expression, The REP-X derivatives are derived from the REP/RIP series above. The original REP/RIP vectors contain an ATG at the 5' end of the polylinker, the X-series removes it and adds a XhoI site. Note that you must supply an ATG on your clone if using the X-series. : the polylinker in the 40-X and 80-X series is not quite the same as the polylinker in the 3X and 4X series. See the map. In pMLtetON, the repressor is co-expressed on the same plasmid. Use of the analogue anhydrotetracycline at 6uM leads to induction within 3 hrs (max 12hrs). Martin Luetzelberger and Erler et al. (2006, Yeast 23: 813–823) tag (from paramyxovirus SV5 p/k protein) fusion, C terminal only right now. Compatible with pSLF172/173 series. Antibody to V5 is commercially available from Invitrogen (R960) or Serotec (MCA1360) N-terminal tag with 3xPk tag (from virus SV5 P-protein) in mid strength (nmt*) nmt vectors with ura4+ or LEU2 markers. Antibody to V5 is commercially available from Invitrogen (R960) or Serotec (MCA1360) C-terminal tag with 3xPk tag (from virus SV5 P-protein) in mid strength (nmt*) nmt vectors with ura4+ or LEU2 markers. Antibody to V5 is commercially available from Invitrogen (R960) or Serotec (MCA1360) used as PCR template followed by yeast transformation for C-terminal tagging of proteins by 3HA, 13Myc, GST, or GFP, respectively, at their normal chromosomal locations used as PCR template followed by yeast transformation for expression of proteins under the nmt1 promoter (3 different strengths) or N-terminal tagging of proteins by 3HA, GST, or GFP, respectively, at their normal chromosomal locations. used as PCR template followed by yeast transformation for expression of proteins under the nmt1 promoter (3 different strengths) or N-terminal tagging of proteins at their normal chromosomal locations. These plasmids allow expression of proteins fused to two halves of YFP, allowing for bimolecular fluoresence complementation in vivo. One putative partner needs to be fused to VN173, and one to VC155. Derived from the REP series, with a lacZ alpha peptide in the polylinker to allow blue/white selection for inserts and GFP fusion. To determine rate of mutagenesis: passage plasmid through pombe and transform into E. coli strain KS40/pKY241 to quantitate System for labeling cells with BudR. One plasmid expresses adh-tk, and one expresses the hENT nucleoside transporter. These plasmids are also available already integrated into the genome of Forsburg lab strain FY2317 Source.


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Last Modified: April 18, 2016 @ 3:08 pm