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

Future Goals

  • Repeat the Construction of Dry Forest Library
  • Optimization of the electroelution step (critical step)
  • Construct a library from Microbial Mats and El Yunque dry forests
  • Plasmid Extraction of the clones that were obtained

DNA Extraction & Library Construction

We decide to do again the library from the beginning. Some changes have been performed to the protocol for the DNA extraction using the Freeze and Thaw method. The soil sample was divide in four bottles and the quantities of the different solutions that were going to be used were readjust. 

Again, we used GITC (guanidine isothiocyanate) instead of Guanidine HCl and because of the lack of Tris buffered phenol chloroform we only used chloroform. After the electroelution we tried to determine the DNA concentration. For each tube we made 3 readings but the values were not consistent and some negative values were obtained.  We decide to use randomly one of the tubes from the electroelution (not the rinse). The DNA was concentrate into a final volume of 100uL but it concentration was not read because of the problems with the other lectures. For the end repair step, 40uL of DNA was taken directly from this tube instead of 40uL of the supposed dilute DNA (0.5ug/uL) from this tube. The other steps of the CopyControlTM Fosmid Library Production Kit protocol were done as directed. Next they, after the inoculation of the possible clones, we can observed some colonies in the plates!!!!

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.

lunes, 2 de mayo de 2011

domingo, 1 de mayo de 2011

February 1, 2011


We decide to work with new samples.

Soil Description:
B #2= sandy, reddish color, abundance of plant material, 45˚C
E #5=slimy soil, brown color, abundance of plant material, 39˚C
C #3=clayey soil, dark brown color, abundance of plant material, 51˚C

At the end of the extraction we decide to realize a master DNA with all of them.  It will help us to create a Metagenomic Library