χρόνος πέρασμα July 13th 2011
- Chlamydomonas reindhartii RNA treated with DNAse to proceed with HydEF and HydG extraction. Obtention of HB101 E. coli to use them as helper cells to conjugate to Rhizobium etli.
- Today Helena Reyes and I cleaned the RNA extraction from Chlamydomonas reindhartii she made from DNA. Here's the mixture we did:
- The mixture was left incubating for 10 minutes at 25°C. After that, 2 μl of EDTA was added to the solution to stop DNAse activity. The solutions were stored at -20°C before doing the electrophoretic gel. Well #1 contains 1kb DNA ladder, Well #2 contains my RNA eppendorf tube treated with DNAse, well #3 contains Helena Reyes's solution. It can be seen that the well I did contains no DNA but little RNA, whereas Helena's contains much RNA but little DNA contamination. Tomorrow we will make cDNA from this RNA.
- As the plasmid pBBRMSC5 has been succesfully standarized, we want to conjugate it to Rhizobium etli to see how it behaves in its final host. The plasmid resides currently in BL21 E. coli cells. We wanted to make S17 E.coli competent cells as these cells have the neccesary components to conjugate plasmids; unfortunetly, the S17 cells we wanted to use aren't working properly, suggesting that they might have lost conjugation capacity through some mutation in one of the proteins needed to conjugate. We will use, instead, a helper cell for conjugation; HB101 E. coli cells that carry the plasmid pRK2013, needed for conjugating. Miguel Ramírez gave us this cells, I passed them to another petri dish with LB and Kanamycin at a concentration of 30 μg/ml. The cells were left incubating at 37°C.