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  • Transformation Protocol

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    Mustafeez M Babar
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    The vector was eluted from paper using NF water and centrifugation, but showed no bands on a 1% agarose gel even after concentrating the sample 10 times. As the nanodrop reading was 1021 ng/uL, transformation was performed directly using the concentrated vector with DH5-alpha E. coli cells, followed by heat shock, incubation, centrifugation, resuspension, and plating on ampicillin plates for overnight growth at 37C.

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    Transformation protocol

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    The vector was eluted from paper using NF water and centrifugation, but showed no bands on a 1% agarose gel even after concentrating the sample 10 times. As the nanodrop reading was 1021 ng/uL, transformation was performed directly using the concentrated vector with DH5-alpha E. coli cells, followed by heat shock, incubation, centrifugation, resuspension, and plating on ampicillin plates for overnight growth at 37C.

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    8 views1 page

    Transformation Protocol

    Uploaded by

    Mustafeez M Babar
    The vector was eluted from paper using NF water and centrifugation, but showed no bands on a 1% agarose gel even after concentrating the sample 10 times. As the nanodrop reading was 1021 ng/uL, transformation was performed directly using the concentrated vector with DH5-alpha E. coli cells, followed by heat shock, incubation, centrifugation, resuspension, and plating on ampicillin plates for overnight growth at 37C.

    Copyright:

    Attribution Non-Commercial (BY-NC)
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    of 1

    1.

    We eluted the vector from the paper by submerging it in NF water and then centrifuging it at
    12000 rpm.
    2. The 1 % agarose gel did not show any bands, of either of the four products.
    3. So, we concentrated the BV (eluted) 10 times (i.e., 100uL were conc. to 10 uL)
    4. Ran it again on 1 % agarose gel, again no bands were observed.
    5. So, it was decided that we should directly go for transformation (as nanodrop was showing a
    conc. of 1021 ng/uL.
    6. Transformation was done using the following protocol;
     10uL of the conc. vector was taken and mixed with 200uL of DH5-alpha E. coli cells
     Chilled on ice for 40 minutes
     Heat shock at 42C for 30 sec
     Chill on ice for 5 minutes
     800uL LB media was added to it and incubated for 2hrs at 37C.
     Centrifuged at 13000 rpm for a minute.
     800uL supernatant was discarded and the rest was resuspended.
     These were then suspended on an Ampicillin containing agar plate and incubated
    overnight at 37C.

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