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I am designing a researching proposal for the class. Because it uses microinjection to up-regulate the gene in C. elegans, the plasmid pCFJ104 - Pmyo-3::mCherry::unc-54 sequences has been chosen. But because the plasmid already has a unc-54, I wonder if there a way that to cut the unc-54 out of the plasmid?

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  • $\begingroup$ Can you give more information on what the unc-54 gene does? Try and be a bit more clear maybe even a picture or link of the plasmid with the unc-54 location, thanks. $\endgroup$ – ATP Oct 31 '15 at 20:48
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    $\begingroup$ unc-54 encodes the major bodywall muscle myosin. In these plasmids the only portion from unc-54 is usually the 3'-UTR and the polyA addition site (AATAAA). myo-3 is either another BWM myosin, or a pharyngeal-specific myosin. This is providing the tissue-specific promoter. The expression plasmid drives expression of the mCherry fluorescent reporter in muscle cells. $\endgroup$ – mdperry Nov 1 '15 at 1:00
  • $\begingroup$ pCFJ104 - Pmyo-3::mCherry::unc-54 is the plasmid I choose, here is the link: addgene.org/19328; because the experiment need to up regulate a gene that is also in the muscle, so before doing the microinjection, the unc-54 need to be cut out before insert the gene I use. So I am wondering how to cut the gene out. Thanks! $\endgroup$ – Scsqpd Nov 1 '15 at 17:50
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Following the link you provided confirms that the C. elegans expression vector will express your insert of choice in the transgenic animals' pharyngeal muscle cells (driven by the myo-3 promoter).

From examining the two images of the plasmid's restriction map on the page you linked to, I infer that a SacI x XbaI double digest should excise the mCherry insert, and be suitable for sub-cloning your fragment of choice.

However you will need to confirm this for yourself. In general, I think it extremely unlikely that you would find an individual on Biology SE who could provide such precise information as "what enzyme sites should I use for my sub cloning experiment."

A general approach would be to

  1. Research to find out if you can obtain a fasta file containing the complete DNA sequence of the starting plasmid.

  2. Search GenBank to see if there is a record containing annotations for the starting plasmid sequence--that will tell you the start points and end points of each functional region.

  3. If the plasmid is not in a sequence database try using the sequence in a BLAST search of a Non-redundant sequence database. In this example you would have retrieved high scoring hits on the unc-54 gene, the myo-3 gene, the mCherry gene, the beta-lactamase gene from pMB9/pBR322/pUC, and the E. coli lacZ gene (among others).

  4. You should also consult the original publication (if there is one) because it may contain additional useful information, including how to contact the authors--who may have sequence files, useful maps, etc.

  5. With the sequence in hand you will be able to create your own restriction map using a suitable software program (there are many commercial ones), e.g. the EMBOSS suite of open source bioinformatics software.

In your case most of this work was done for you because the AddGene site had two annotated restriction maps that showed almost all of the information you needed. N.B. One map had the locations of the useful restriction enzyme sites, and the other map had the annotations showing the extent and locations of the worm fragments and the reporter gene. It was only by integrating the information from both maps that I was able to suggest one possible solution to your query.

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