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Thursday, March 1

  1. 8:58 am

Friday, May 28

  1. page RNAi Lab edited ... Feeding Transformed HT115 to Worms - Dr. Jeremy M. Rosenblatt The man (or boy wonder) in cont…
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    Feeding Transformed HT115 to Worms - Dr. Jeremy M. Rosenblatt
    The man (or boy wonder) in control of all of these scientific publications is Mr. Eric J. Jepeal, MBA.
    {IMG_6057.JPG}
    From left to right: Dr. Radocchia, Lord Jepeal, Dr. Bacay, Ms. Appel, Dr. Lee, Dr. Rosenblatt, Dr. Nightingale, and Dr. DeRogatis

    Want to know more about us? Meet the ones controlling the world of the C. elegans here.
    For more information regarding C. elegans and specific mutations, please click here.
    (view changes)
    5:29 pm
  2. file IMG_6057.JPG uploaded
    5:28 pm
  3. page The Creation of the Cloning Vector Using L4440 edited ... Why use L4440? {vector.jpg} ... Conveniently located for controlled to control the ex…
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    Why use L4440?
    {vector.jpg}
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    Conveniently located for controlledto control the expression of
    ...
    one another, thus closingand thus, the plasmid would close before it
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    our C. elegans.elegans!
    Procedure and Results
    1. 2 cultures are needed in order to make four separate mini-preps:
    (view changes)
    1:01 pm
  4. page The Creation of the Cloning Vector Using L4440 edited ... Creation of the Cloning Vector Using L4440 What is a cloning vector? ... copies of the a…
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    Creation of the Cloning Vector Using L4440
    What is a cloning vector?
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    copies of thean inserted piece
    Why use L4440?
    {vector.jpg}
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    interest, Blister-1. Conveniently, in L4440,Conveniently located for controlled expression of our target DNA, the MCS is flanked by two T7
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    the bacteriophage T7.T7, flank the MCS. By choosing
    ...
    the PCR product.product, which should contain our gene of interest. For our
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    PCR product. EffectiveChoosing two different enzymes to cut within the T7 promoter was key. Using only one restriction enzyme to digest the L4440 vector would have created a higher probability of the sticky ends annealing to one another, thus closing the plasmid before it could receive the gene of interest.
    Effective
    ligation of
    ...
    of DH5alpha cells.
    Procedure
    cells and will bring us one step closer to gene silencing in our C. elegans.
    Procedure and Results

    1. 2 cultures are needed in order to make
    LB tubes
    AMP (1,000 x) 1 uL/mL
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    5 ul NEB Buffer #2 (Click on the link to be directed to the NEB website double digest finder for HindIII and SacII).
    10 ul ddH2O
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    confirm that the four mini-preps and the digestion of
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    were successful.
    {AFB_Mini_Prep_Gel_1_1.jpeg}

    {ABPicture_1.png}

    6. Digest the PCR product using the following protocol. Place the digestion tube in a 37 degree Celsius water bath for several hours.
    20 ul Bli-1 PCR product
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    3 ul NEB Buffer #2
    2 ul ddH20
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    to verify successfulthat the digestion of PCR productwas successful and properly functioningthat HindIII and SacII.SacII are functioning properly (i.e. cutting).
    {PCR_L4440_Digest_Gel-2.jpeg}
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    restriction enzymes in theseby placing the two tubes. Place the restriction enzymestubes in a
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    the following protocols.protocol.
    16 ul digested L4440 plasmid
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    PCR product (should contain multiple copies of our gene of interest, Blister-1)
    2 ul ddH2O
    2 ul T4 ligase
    (view changes)
    12:57 pm
  5. file ABPicture_1.png uploaded
    12:34 pm
  6. page The Creation of the Cloning Vector Using L4440 edited ... In our RNAi experiment, use of the L4440 cloning vector (pictured above) will hopefully result…
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    In our RNAi experiment, use of the L4440 cloning vector (pictured above) will hopefully result in successful recombinaton of the plasmid and our gene of interest, Blister-1. Conveniently, in L4440, the MCS is flanked by two T7 promoter sequences, which originate from the bacteriophage T7. By choosing two enzymes that are compatible in a double digest and that both have restriction sites in the MCS, we can open up the L4440 plasmid, thereby preparing it for ligation with the PCR product. For our purposes, we have chosen restriction enzymes HindIII and SacII to digest both our cloning vector and our PCR product. Effective ligation of the L4440 cloning vector and the Blister-1 gene will allow for the subsequent transformation of DH5alpha cells.
    Procedure
    ...
    to make four separate mini-preps:
    LB tubes
    AMP (1,000 x) 1 uL/mL
    ~500 uL L4440 (in DH5alpha) per culture
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    in a 37 degree Celsius shaking water
    3. Conduct 4 Zippy mini-preps to separate the L4440 plasmid from the E. coli DH5alpha strain host cells.
    4. Run a gel to confirm that mini-preps were successful.
    {AFB_Mini_Prep_Gel_1_1.jpeg}
    5.
    Digest the
    ...
    following protocols. Place in a 37 degree Celsius water bath for several hours.
    30 ul L4440 plasmid
    2.5 ul HindIII
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    5 ul NEB Buffer #2 (Click on the link to be directed to the NEB website double digest finder for HindIII and SacII).
    10 ul ddH2O
    6.5. Run a gel to confirm that mini-preps and digestion of L4440 were successful.
    {AFB_Mini_Prep_Gel_1_1.jpeg}
    6.
    Digest the
    ...
    the following protocols.protocol. Place the digestion tube in a 37 degree Celsius water bath for several hours.
    20 ul Bli-1 PCR product
    2 ul HindIII
    ...
    3 ul NEB Buffer #2
    2 ul ddH20
    7. Put both products from Steps 4 and 5 into a 37 degrees Celsius water bath.
    8. After the two digests have remained in the water bath for a few hours, run
    Run a gel
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    verify successful digestion. Lanes should consistdigestion of digestedPCR product and undigested L4440 plasmid, digestedproperly functioning HindIII and undigested, PCR product, a 1 kb ladder, and a 100 bp ladder.SacII.
    {PCR_L4440_Digest_Gel-2.jpeg}
    9.8. If both digestions were successful, combinethe L4440 plasmid and the PCR product have been digested properly at this point, denature the restriction enzymes in these two tubes. Place the restriction enzymes in a 65 degree heat block for 20 minutes.
    9. Combine
    the two separate digests in a
    16 ul digested L4440 plasmid
    16 ul digested PCR product (should contain multiple copies of our gene of interest, Blister-1)
    (view changes)
    12:34 pm
  7. page Caring for C. elegans edited ... NGM-lite Plates Recipe for NGM-lite Plates: This recipe makes enough solution for about …
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    NGM-lite Plates
    Recipe for NGM-lite Plates:
    This recipe makes enough solution for about 35 plates. Now, since one can mess up plates by either contaminating them or allowing the OP50 to run to the edge of the plate, the recipe usually yields 30 pristine plates. Mistakes happen.
    (view changes)
    11:04 am
  8. page Caring for C. elegans edited ... Transferring worms from NGM-lite plate to NGM-lite plate: Chunking Touching the sides of the …
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    Transferring worms from NGM-lite plate to NGM-lite plate: Chunking
    Touching the sides of the plate jeopardizes the sanitary integrity of the C. elegans environment. Transferring the worms onto 'fresher' plates is crucial for preventing the worms from dying whether they run out of their food source, OP50, and starve, or the NGM-lite medium dries out. An easy and effective way to transfer the worms, and the way that I have transferred the worms, is called "chunking." Chunking entails dipping a scalpel in alcohol and burning off the excess alcohol. Once the scalpel is cooled off, cut a chunk from the NGM- lite medium about 1 cm square and flip the chunk over unto a seeded plate. Simply enough, the worms crawl out from the chunk onto the 'fresh' plate. The N2 strain of worms have now been transferred effectively. After about every 3-5 days, I repeat the chunking process onto seeded plates. One must be sure to put the seeded plates, being stored in the refrigerator, into the incubator, making sure the medium is near room temperature.
    Healthy Worms:
    {fri78N2worms.jpg}

    A Very Informative Source:
    http://www.wormbook.org/chapters/www_strainmaintain/strainmaintain.html
    (view changes)
    11:02 am

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