We will be working with up to four plasmids: pJW1219, pJW1583, pJW1585 and pSEC103. The pJW plasmids are a gift from Dr. Jordan Ward now at UC Santa Cruz and pSEC103 is a gift from Dr. Thomas Boulin at the University of Lyon.
pJW1219 contains Cas9 and the sgRNA (lacking the guide sequence). sgRNA=single guide RNA. eft-3p is a worm promoter that is designed to drive expression of Cas9 in germ cells. NLS=nuclear localization signal. U6p, a RNA polymerase III promoter, is designed to transcribe sgRNA so that it remains in the nucleus of cells. We will use site directed mutagenesis to insert our guide sequence into this plasmid.
pJW1583, pJW1585 and pSEC103 will be used to create our repair templates. SEC=self-exciting cassette (the portion of the plasmid that will be removed in worms upon heat shock). loxP = a site that can undergo homologous recombination with another loxP site (not shown as it is present in the SEC) when Cre Recombinase is expressed. ccdB = a suicide gene. Plasmids harboring this CDS can only grow in survival cells. AID= auxin inducible degron. 3X FLAG and MYC are protein tags. pJW1583 and pJW1585 will be either cut with SpeI/Avr II (to create C-terminal knock-ins) or Spe I/ClaI (to create N-terminal knock-ins).
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