User talk:Nkuldell/mtDNA pt3: Difference between revisions

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*2. removed EcoRI site from coding sequence of RNT1. Changed GA<b>A</b>(bp721-723)TTC(bp724-726) at Glu240Phe241 to GA<b>G</b> TTC which removed RI but keeps GluPhe.<br>
*2. removed EcoRI site from coding sequence of RNT1. Changed GA<b>A</b>(bp721-723)TTC(bp724-726) at Glu240Phe241 to GA<b>G</b> TTC which removed RI but keeps GluPhe.<br>
**[[Media:Macintosh HD-Users-nkuldell-Desktop-RNT1 genomic noRI(baseA723G).rtf| sequence file]] for RNT1 genomic DNA with silent mutation to remove EcoRI
**[[Media:Macintosh HD-Users-nkuldell-Desktop-RNT1 genomic noRI(baseA723G).rtf| sequence file]] for RNT1 genomic DNA with silent mutation to remove EcoRI
[[Image:Macintosh HD-Users-nkuldell-Desktop-RNT1 genomic noEcoRI(A723G).PNG.PNG|thumb| center| RNTI with silent mutations to remove EcoRI; restriction with favorite enzymes]]
**Favorite Enzymes: AvaI, BamHI, BglII, EcoRI, HincII, HindIII, PstI, SmaI, XbaI, XmnI

Revision as of 12:19, 26 June 2007

Experimental Details

deoxycycline-regulation of RNase expression

plasmid map for pCM224
  • regulation of expression from deoxycline-induced promoter, described in Herrero Yeast 1998 14(12):1127-38
  • sequence file for pCM224 which encodes tTA and CYC1-repressible promoter due to 2 copies of tetO
  • can excise relevant regulatory module (tTA driven by ADH1 and CYC1 with 2XtetO) on RI, BamHI fragment

pRS416

plasmid map for pRS416 with single cutters indicated
  • sequence file for pRS416
  • can clone tTA/CYC1tetO2/mtRNT1 into RI, BamHI bkb

mtRnt1

Steps to target RNT1 to mitochondrial matrix

  • 1. downloaded genomic sequence of RNT1 from SGD. RNT1/YMR239C on chromosome XIII from coordinates 749676 to 748261 on Crick (bottom) strand.
  • 2. removed EcoRI site from coding sequence of RNT1. Changed GAA(bp721-723)TTC(bp724-726) at Glu240Phe241 to GAG TTC which removed RI but keeps GluPhe.
    • sequence file for RNT1 genomic DNA with silent mutation to remove EcoRI
RNTI with silent mutations to remove EcoRI; restriction with favorite enzymes
    • Favorite Enzymes: AvaI, BamHI, BglII, EcoRI, HincII, HindIII, PstI, SmaI, XbaI, XmnI
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