PowerPoint Presentation - Recombinant DNA Bacterial Transformation-Student Instructions Plasmid Digest

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Recombinant DNA Bacterial Transformation Student
Instructions Plasmid Digest
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Recombinant Transformation Plasmid Digest

• Obtain four 1.5 ml reagent tubes labeled
• - pAmp
• - pKan
• - 2XRB(Restriction Buffer)
• - B/H (BamHI and HindIII
• enzymes).

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Recombinant Transformation Plasmid Digest
Use the matrix below as a checklist while adding
reagents to the pAmp and pKan tube. Use a fresh
pipette tip for each reagent. Add all reagents
directly to the solution.
NOTE The buffer is always added before the
enzymes. Tube 2X RB B/H _________
_________________________________________________
pAmp 7.5 µl 2 µl pKan 7.5
µl 2 µl
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Recombinant Transformation Plasmid Digest

• Place the reaction tubes in a 37oC water bath and
  incubate for 30-60 minutes.

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Recombinant Transformation Plasmid Digest

• After incubation, freeze the reaction at -20oC
  until you are ready to proceed to Ligation. You
  can store reactions at this temperature for
  several days.