User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/08

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Ligation control for blunting products

ABSTRACT

Experiment that proves if the blunting experiment was successful. Ligation of the blunting products and transformation.

  • It was scheduled for today that we would clean the products of the blunting experiment, Melissa an I went to see Miguel in the morning and we showed him the picture of the agarose gel that Daniel did yesterday. He told us that because of the little amount of DNA that was observed in the gel, the best thing to do was not to clean the DNA and perform a control to corroborate that the blunting was successful. For this purpose we ligated the blunting products, the idea is that if the blunting was well performed and the ends are blunt, the ligation reaction should circularize the plasmid, then with this plasmids we could transform bacteria and see if colonies are formed. It should be noted that if there are not many colonies means that the DNA does not work for our purposes of standardization and we would have to start all the process again. So lift your thumbs up!!

The ligation was performed with the following reagents concentrations:

Reagent Quantity
H20 24μL
Blunting DNA 2μL
Buffer T4 10x 3μL
T4 DNA ligase 1μL

We made two reactions in two different tubes (labeled 1 and 2). Once we have mixed everything, we centrifuge the tubes for 20 seconds. Then we incubated the tubes at 25 degrees. We started the experiment around 10 am, so we have to transform at 8 pm.

Transformation

  • For the transformation we follow the protocol that Miguel gave us. We use the tube 2 of ligation reaction. Because we could not enter the laboratory of Dr. Romero to pick up our competent cells, Uriel gave us cells, two tubes of 100μL.
  • Pablo made 4 petri dishes:
  1)Plated with 100μL of the product of transformation directly. Many colonies expected.
  2)Plated with the resuspended pellet resulting from centrifuging the product of the transformation. Many colonies expected. This was done to obtain a major concentration of bacteria.
  3)Plated with the control of transformation. No DNA was used for the transformation. No colonies expected
  4)The plating control. No bacteria added to the plate. No colonies expected.

Seed surface disinfection

ABSTRACT

Preparation of the Phaseolus vulgaris seeds for germination by a surface disinfection.

  • Today I started with the gardening activities. The first step was to prepare the seeds of Phaseolus vulgaris for germination.
  • The following protocol has to be done:
  1)Select and collect in a petri dish the seeds of the plant of interest.
  2)Wash  the seeds with regular water. Put some water in the petri dish, then shake it for a few seconds and remove the water. You can do this step twice if the seeds are very dusty.
  3)Put the seeds in an Erlenmeyer flask. Add 70% ethanol in a volume covering the seeds an let it stand for 5 minutes.
  4)Remove the ethanol.
  5)Add to the flask 25% chlorine in a volume covering the seeds and let it stand for 15 min.
  6)Remove the chlorine.
  7)Wash the seeds 8 to 10 times with sterile water, until no chlorine odor can be detected.

Along with seed disinfection we prepare a solution of bacteriologic agar at 0.75% in H2O. We sterilize the solution in an autoclave for 1 hour at 0.5 MPA. When the solution was cold Pablo put it in 5 petri dishes.


Daniel managed to place about 20 seeds in each petri dish, and we left them incubating at 30°.


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