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From 2014hs.igem.org

April 18th, 2014

Alli Ambrosini

Did research on how we will be testing the results of our project in class today. We will need to get non-nutrient agar plates so that we can test whether the bacteria can eat our PHB at all. Normal agar has dextrose in it, which bacteria will consume before they touch PHB because its easier to eat. A simple sugar is a lot easier to break down than a long polymer such as PHB.

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Lauren Ewell, John Grammer

We took competent cells, centrifuged them to get a pellet. We resuspended in Luria broth, centrifuged and then we combined into three tubes and added calcium chloride and put on ice for 20 minutes. Then Alli took over after that step. She centrifuged gently by pulsing 10 times and then resuspended with 200uL of cold calcium chloride.

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Alex Manchester, Nick Keen, Anders Beaurline, Jessica Prax, Bailey Fernandez

Did the re-suspending of DNA and transformation procedure.

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.

2. Pipette 10uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully re-suspended.

3. Transform 5uL of the resuspended DNA into your competent cells, plate your transformation with the appropriate antibiotic* and grow overnight.

4. We are going to use the chloramphenicol plates. Be sure to label them.

We weren’t sure the cells were competent because they weren’t suspended, there was a pellet at the bottom

We then did the transformation procedure in the iGEM procedure notebook on page 28. We did not have 2 hours to incubate it, which was step 6

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