Reverse Parts with PCR

From 2006.igem.org

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Revision as of 11:50, 21 June 2006

Dissolving PCR primers

  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 DNA template

DNA template: Add 15 ul dH2O to appropriate well on plate DNA-1 or DNA-2. Take 1 ul of this and add it to 20 ul water and label as "1/20" then part number. Use 1 ul of this in PCR.

After PCR, 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.

Restriction digest as follows:

these two things in separate tubes with both XbaI and SpeI

  1. Band from PCR
  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, along with the hix ligations (take out one tube of 200 ul cells and divide it among the transformations, probably 200/6 = 33 ul each)