User:Paulina Alatriste/Notebook/UNAM Genomics Mexico 2011/2011/06/08
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Ligation control for blunting products
Experiment that proves if the blunting experiment was successful. Ligation of the blunting products and transformation.
The ligation was performed with the following reagents concentrations:
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.
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
Preparation of the Phaseolus vulgaris seeds for germination by a surface disinfection.
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.