Lab July 22 2009

From 2009.igem.org

Doing the test of competence today.

Here's the protocol from openwetware: Measurement of competence

   * Transform 50 μl of cells with 1 μl of standard pUC19 plasmid (Invitrogen)
         o This is at 10 pg/μl or 10-5 μg/μl
         o This can be made by diluting 1 μl of NEB pUC19 plasmid (1 μg/μl, NEB part number N3401S) into 100 ml of TE
   * Hold on ice 0.5 hours
   * Heat shock 60 sec at 42C
   * Add 250 μl SOC
   * Incubate at 37 C for 1 hour in 2 ml centrifuge tubes rotated
         o using 2ml centrifuge tubes for transformation and regrowth works well because the small volumes flow well when rotated, increasing aeration.
         o For our plasmids (pSB1AC3, pSB1AT3) which are chloramphenicol and tetracycline resistant, we find growing for 2 hours yields many more colonies
         o Ampicillin and kanamycin appear to do fine with 1 hour growth
   * Plate 20 μl on AMP plates using sterile 3.5 mm glass beads
         o Good cells should yield around 100 - 400 colonies
         o Transformation efficiency is (dilution factor=15) x colony count x 105/µgDNA
         o We expect that the transformation efficiency should be between 5x108 and 5x109 cfu/µgDNA

There are some questions in this protocol, and some places that we have deviated slightly from it.

First, we could not find any pUC19, but pUC18 was available. Since we are using only the ampicillin resistance from the plasmid to test competence this should not be problematic.

Second, our pUC18 was at an initial concentration of 230 ng/ul, so to get to our desired concentration of 10 pg/ul we diluted 0.5 ul of pUC18 into 11.5ml of TE. We stored 3 ml of this diluted pUC18 and have enough original stock for several more dilutions if needed.

The temperature of the SOC was not noted in the protocol. We had stored this in the 4 degree fridge, so that is the temperature we used it at.

We did not have any 2ml centrifuge tubes, and the BIOC301 instructors have a bit different transformation protocol using glass test tubes instead of microfuge tubes. Apparently the time in the heat shock and the agitation are both different, so we followed the same protocol as above, but in 13mm glass test tubes rather than a microfuge tube.

Finally, we did not have glass beads for the final spread plate step, so we made the spread plate using our normal sterile glass spreader technique.

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