Making Reverse pARA

From 2006.igem.org

Latest revision as of 11:58, 21 June 2006

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)

Restriction digest as follows:

  1. 20 ul eluted PCR product or pTet-GFP
  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

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)