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!!!!