Reverse Parts with PCR

From 2006.igem.org

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:

  1. 20 ul eluted PCR product
  2. 16 ul dH2O
  3. 2 ul NEB2 10X buffer
  4. 1 ul XbaI
  5. 1 ul SpeI

Heat inactivate the restriction enzymes by putting the tubes at 65C for 20 minutes

Set up ligations

  1. 20 ng pTet-GFP X/S only
  2. 20 ng pTet-GFP X/S + 5 ul X/S digest from above

Transform JM109 cells (take out one tube of 200 ul cells and divide it among the transformations)