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From 2011.igem.org

The Lab Opens Today at 5:00 pm. Could someone please email our googlegroup to this effect? For some reason I am unable to sign into my GMail or TU accounts!. Dr Liz

The time the Lab will this Thursday will depend upon the responses to this request: Please add your name and the time you intend to come to the lab on this day. Put an asterisk(*) before your line to add it to the list. Tomorrow at 1:00pm I will check this page, and the lab will open at the earliest time that at least 2 iGEMers intend to arrive. An email to the googlegroup will be sent, and the opening time, although it should be obvious to all, will be posted at the top of this page.

1. On 8/4/11 we performed colony PCR on the two colonies (one from 3B and one from 4A) that we obtained by transformation on 7/31/11. Today we are running a gel of the PCR products that were amplified by colony PCR on 8/4/11. We prepared 1 % gel and did the colony PCR from 3B and 4A.

Gel Electrophoresis of PCR Reaction 8.4.11 We used 6 lanes and ran 25 ul of PCR products (except for the bench top ladder):

• Lanes 1 and 6 are 6 ul of Bench top ladder.(600 ul 100lanes, 1kb)

• Lane 2 -ve control (is this positive control?)

• Lane 3 3B

• Lane 4 4A

• Lane 5 No template

The gel was started at 9.14 at 82 volt and stopped at 10.10.

Rocking started at 10.10 at 50 rpm.

2. We checked the results of the transformation from 8/4/11. Results:

• Negative control: no colonies
• 3B undiluted: 9 colonies
• 3B diluted 1:10: 2 colonies
• 4A (plate says from 50 ul of transformation): >100 colonies
• 4A undiluted: 3 colonies

We set up colony PCR from these colonies in case none of the colonies from the 8/4/11 colony PCR are correct.

Colony PCR was done by taking sterilized toothpicks and very lightly scratching the surface of the colonies one time, while simultaneously turning the toothpick along its axis. For the case of 3B and 4A undiluted colony plates, the colonies were marked on the outside of the plates. These were then directly dipped vigorously into PCR mix (no dilution). 4A plate with > 100 colonies had randomly selected separate colonies scratched similarly by toothpicks, but these were then very lightly scratched onto a new colony plate, with the scratches indexed with markings on the outside of the plate (this was done because there were so many colonies that it would have been difficult to distinguich which colony was which on the original plate) The toothpick for the particular colony was then dipped into alliquoted PCR mix (each sample having a total volume of 50 ul. PCR mix used was same as recipe shown on August 4th notebook page, except that mastermix is 25x, (rather than the 5x shown)

After these PCR reactions are completed, they need to be run on a gel and then, if there is product, cut with PstI. We ran 24 PCR reaction (a negative control with no template DNA, a positive control using the pET-Taq plasmid as a template, and 22 reactions using bacteria from the plates as templates).

3. We also checked the results of the transformation of Biobrick parts from the registry from 8/4/11. We had no colonies for either the part from the 5I or 5K wells. Dr. Burkett will work on repeating the transformation in his lab in case there was a problem with the cells or the antibiotics in the plates.

Person/ Time

• Dr Liz Time = to be determined
• Travis; approximately 5pm
• Steve: 7:15 to 10:30 PM
• Bivor: From 12 and to 10.30pm
• Desirae: 2-10:30pm