Creating a TetR Transposon

Made freezer stocks of brothed cultures:
                      200 uL of 80% glycerol + 800 uL of broth culture

         Stocks of pUC19 in LBAmp100, pBE110 in LBTet15, pEx18Tc in LBTet15
         Stored in EW/Purple Box 2 in the -80 C

Spread plated 100 uL of pBE100, pBE110, pTnMod-Okm, and pEx18Tc onto LBKan100 to confirm that the KanR gene was really removed in the pBE110 and was still present in pBE100. The other two plasmids serve as controls. 100 uL of broth culture spread onto each plate.

Made crude genomic lysate of pBE100, pBE110, pTnMod-Okm, and pEx18Tc.

1. 40 uL ddH20 + isolated bacteria colony from agar plate
2. Run thermal cycler "Lysate": 55 C for 10 minutes
                                                  80 C for 10 minutes
                                                  20 C for infiniti
3. Added 80 uL of ddH20
5. Briefly centrifuge for 30 seconds at 5000 rpm to pellet

Note: No Proteinase K was used. This method should only be used to create DNA templates for less sensitive procedures such as PCR. Only use supernatant of crude genomic DNA and avoid disturbing pellet.

Amplified tetracycline resistance gene in pBE100, pBE110, pTnMod-Okm, and pEx18Tc using the primers mentioned in the 5-18-14 post (ones with the restriction enzyme flanking regions).

            20 uL PCR SuperMix
            1 uL Forward Primer
            1uL Reverse Primer
       +   3 uL Template DNA
            25 uL Total Volume

PCR parameters:
     1. 95C 2 minutes
     2. 95C 1 minute
     3. 60C 1 minute
    4. 72C 2 minute
    5. Repeat steps 2-4 25 times
    6. 72C 5 minutes
    7 20C infinitely

Amplicons were stored at -20 C

//EWW