OBJECTIVES:

The objective of this blog is to provide updates about what we are realizing at lab. Our work will be to repeat the procedures that we had learn in our extraordinary experience at Yale in the Mastering Metagenomics Workshop in 2010 Summer.

UPRM Team 2010

UPRM Team 2010

lunes, 23 de mayo de 2011

Library Construction

For the library construction we used the DNA extracted from the electroelution that was preserved at -20oC. DNA concentration was measured again with the spectrophotometer to verify if we had DNA. The obtained DNA concentrations were different from the last time we measure it. The concentrations obtained were 15, 190, 55 and 3.3ng/uL for the tubes of 1, 1R, 2, and 2R respectively. It was strange, the fact that in R2 (R2 stands for the rinse of the DNA obtained after the electroelution of noodle 2) the concentration of DNA was greater than tube 1 and very similar to tube 2.  For the Library construction we decide to use 2R. Before the library construction we concentrate the DNA obtaining a final DNA concentration of 1350ng/uL. After this, we proceed with the library construction following the instructions of the CopyControlTM Fosmid Library Production Kit. For the growing of the possible infected cells or clones we use LB agar with 12.5ug/mL of chloramphenicol and we did it too with a lower chloramphenicol concentration of 6.5ug/mL (were made by mistake). We growth the EPI300 (not resistant to cloramphenicol) in 6.5ug/mL of chloramphenicol to verify if it was capable of growth but it was not.  The plates in which the possible clones were infected with non dilute phages and the dilution of 1:10 show some colonies growth, but it was only in the plates of the lower concentration of chloramphenicol (6.5ug/mL). To verify the chloramphenicol resistance we inoculate the possible clones in 12.5ug/mL of chloramphenicol and were incubate overnight. Not all the transferred colonies growth. The growth ones were preserved and a plasmid extraction is going to be performed to confirm and see if it has the vector and the DNA insert.

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