Making Reverse pARA

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PCR primers arrive Tuesday

  1. spin down tubes with dry DNA
  2. calculate volume dH2O to give 200 pmol/ul, label as "10X"
  3. dilute 10X to make 1X

Conduct PCR (30 cycles, use Tm to determine annealing temp)

  1. 1 ul Forward primer
  2. 1 ul Reverse primer
  3. 2 ul 10X with no Mg
  4. 2 ul 25 mM MgCl2
  5. 12 ul dH2O
  6. 1 ul Taq
  7. 1 ul plasmid with pARA (labeled I13453, in right freezer)

Add 5 ul 5X loading buffer and run on 7% PA gel (product should be 150 bp)

If this works, scale up the PCR to 5 x 20 ul, run new PA gel, and purify band. Elute in 2 x 40 ul.

Digest these two things in separate tubes with both XbaI and SpeI

  1. 150 bp pAra band from above
  2. plasmid I13522 (pTet-GFP, glows green with UV)

Design and set up ligations

  1. 20 ng pTet-GFP X/S only
  2. 20 ng pTet-GFP X/S + 10 ng pARA X/S

Transform JM109 cells

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