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    DIY Biology Project - Splash 2022
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    DIY Biology Project - Splash 2022

    Friday, 11/18/2022Designing our plasmid sequenceGetting the sequence components:Plasmid backbone - pUC19 amilCP sequences - mUAVPutting the amilCP sequence into pUC19 using restriction enzymesBasic idea: we will use a restriction enzyme to cut both pUC19 and amilCP, and then glue the amilCP into pUC19Which restriction enzyme?Adding HindIII sites to our amilCP using custom primers2. Putting the plasmid in our bacteria
    Friday, 11/18/2022
    1.
    Designing our plasmid sequence
    Getting the sequence components:
    Plasmid backbone - pUC19
    amilCP sequences - mUAV
    https://www.addgene.org/102680/
    Putting the amilCP sequence into pUC19 using restriction enzymes
    Basic idea: we will use a restriction enzyme to cut both pUC19 and amilCP, and then glue the amilCP into pUC19
    Which restriction enzyme?
    We want to insert our gene next to the promoter in pUC19, so we should use some of the sites in the pUC19 multiple cloning site
    But, it can't cut anywhere else in pUC19 or it will mess up the rest of the plasmid
    It also must not cut anywhere inside our amilCP sequence or we won't get the full protein in
    Let's use the Benchling digest tool to check.
    Is HindIII OK?
    *We are also going to use the ATG site from pUC19, so we have to make sure when we add amilCP, it will still be in-frame
    Adding HindIII sites to our amilCP using custom primers
    We will need to use PCR and design primers to amplify amilCP with the HindIII sites added, and enough room for the enzyme to actually cut the amilCP
    From NEB:
    https://www.neb.com/tools-and-resources/usage-guidelines/cleavage-close-to-the-end-of-dna-fragments
    It looks like anything more than 3 basepairs should be enough
    Making the primers and then ordering them!
    -Usually, I try around 20-30 basepairs to start
    https://www.idtdna.com/calc/analyzer
    Look for PCR primers that conform to the following guidelines (use our free online OligoAnalyzer® tool for this purpose):-
    > The difference between melting temperatures (Tm) of the primers should be less than 5°C.
    -> The GC content should be between 35-80% or equivalent to the product being amplified.
    -> The Delta G value of any self-dimers, hairpins, and heterodimers should be weaker (more positive) than -9.0 kcal/mole. Positive numbers indicate that the actual secondary structure shown will not form at all.
    -> Avoid 3' complementarity between the two primers to prevent primer dimers.
    Do the PCR...
    template + nucleotides + primers + buffer + polymerase enzyme
    thermocycler
    Do the digest...
    template + restriction enzyme + buffer
    heat block
    Do the gel and purify your fragment...
    Gluing together the amilCP and pUC19 to make a plasmid
    amilCP fragment + pUC19 fragment + T4 DNA ligase
    2. Putting the plasmid in our bacteria
    Let's go back to our slides
    https://docs.google.com/presentation/d/1P0ltBV6BYnad98J4JGC8qPdxlBeCJD0nb0v1QY7-uNs/edit#slide=id.g192d14a4de9_0_14

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