From 2007.igem.org

Work Progress

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23/07/07 - Our First Day

The first challenge: Prepare all the necessary to start the wet lab.

Objectives: Today's objective is prepare all the plates we are going to need in the project.

Method: The first step was to make three liters of the medium and sterilize it with the autoclave. After an hour and a half, (and a little snack), we add the antibiotics and let the medium get cold. With the medium, we do 160 petri plates and then we keep it in the refrigerator.

We took some bacteria and put it in and LB media and let them grow at 37ºC all night.

24/07/07

• The overnight culture was glycerinated at a final concetration of 30% glycerol.
• We surfed the registry so to order mentally what we're going to transform tomorrow. The information about the parts is:

Name	Part	Plate	Well
Promoter lacI	R0011	1	7M
Promoter tetR	R0040	1	7O
lacI repressor	C0012	1	5A
tetR repressor	C0040	1	5C

25/07/07

Objectives: Today is an important day, because we must make a transformation with the chosen parts.

Method: We are going to transform the plasmids. This is the protocol we have used. See more details in Transformation Protocol

• We dilute the plasmid with 15 μl of H20 in the well and then we store it into a pcr tube.
• We take 1 μl of the chosen plasmid and we mix it with 100 μl of chemically competent bacteria.
• We use a heat shock protocol with the following steps:
  • 30 minutes in ice
  • 30 seconds at 42ºC (45 sec is also used)
  • 5 minutes in ice
• Then we put in 900 μl of LB media and leave it at 37ºC during an hour.
• After the hour we centrifuge it at 3000 rpm during 10 minutes (our centrifuge has a little radius, else we would do it at 1000 rpm).
• We take the supernatant away, about 900 μl, and resuspend the pellet with the 100 μl left.
• The last step is to plate it in a Petri dish and incubate it overnight at 37ºC.

We have four plates for our four plasmids...

26/07/07

After a night we discover that only the plate with pLacI has grown. 1 out of 4 is a good result? Some minutes later we decide to try it again with the other three plasmids. We must repeat all the process again... We pick three colonies of the pLacI and we put it in LB media with ampicillin. We keep it over night at 37ºC with the aim to make it grow.

27/07/07

Again the same result (now 1 out of 3); just two colonies in the ptetR plate have grown. We put glycerinate the three tubes of pLac and keep them at -80ºC. (why three? just to be sure to have some colonies that are pLacI)

• We miniprep the rest of the overnight culture, but the yield of it is so poor (15 ng/μl) that we have to repeat it.

30/07/07

We are going to transform the other two plasmids, using another strain: DH5α We repeat the same protocol like the last days.

• We inoculate the glycerinated pLacI and both colonies from the pTetR plate into fresh LB media and leave it o/n; so we will be doing minipreps tomorrow(!).

31/07/07

There is nothing in the plates...(why?) We repeat the same transformations with lacI and tetR, using DH5α strain. We do the minipreps of the ptet and plac. Our results: there aren´t plasmids in our bacterias.

After the lunch, we repeat all that we did this morning. We hope to get something tomorrow...

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