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P-613 CRY1 marker; RPS14 gene pJN4

Name: P-613 CRY1 marker; RPS14 gene pJN4
SKU: P-613
Availability: InStock

$30.00

From Pete Lefebvre, 1994

CRY1 marker; RPS14 gene, ca. 4 kb EcoRI fragment in pUC119

Derived by the following steps: pCRY1-1 was constructed by using PCR to amplify the 3.6 kb genomic region containing the entire CRY1-1 transcription unit and 250 bp of upstream sequence. The 3.6 kb product was cloned into pUC119 digested with XbaI and SacI. Plasmid pRS2-1 was constructed by amplifying a 1073 bp region of the RBCS2 promoter and cloning the PCR product into pUC119 deigested with SmaI and dT tailed with Taq polymerase. pJN1 was constructed by inserting the 3.0 kb BfrI-EcoRI fragment of pCRY1-1 into the HincII site of pRS2-1. pJN4 was constructed by ligating the 474 bp Alw26I-ApaI fragment of pCRY1-1 to the 7.2 kb ApaI-BamHI fragment of pJN1.

Lefebvre included the following suggestions on use of this plasmid:

The protocol we use for transformation is included in the Nelson et al. paper. As you will notice in the paper, the RBCS2 promoter was deleted and inverted during the PCR steps of constructing pJN4, but it still works well for transformation.

The key point to be stressed is the need to determine the appropriate killing concentration for emetine for your strain under your conditions. Emetine is variable in potency between batches, and it is light sensitive. You should determine, by replica plating, the killing concentration for your strain, and then attempt your selection on a concentration 2-3x that needed to kill all of your wild-type. We grow our cells on plates in the light, but not very strong light (about 15 inches away from a two-bulb fluorescent shop light). Make your plates only a few days in advance, and keep in the dark until use.

You might want to try selection in minimal media as well as acetate-containing media. Expression from the RBCS2 promoter is maximal in light in media without acetate.

host strain: DH4 alpha
amp resistant

Nelson JA, Savereide PB, Lefebvre PA (1994) The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation. Mol Cell Biol 14:4011-4019

P-613 CRY1 marker; RPS14 gene pJN4

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Banner Photo Credit: Chlamydomonas reinhardtii zygotes were stained for immunofluorescence with anti-acetylated tubulin (green), anti-phospholipase D (red), and DAPI (blue) by Karl F. Lechtreck (University of Georgia) and George B. Witman (University of Massachusetts Medical School).

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