This past weeks I have been working on the periplasmic experiments (details on my February 04 post). This experiments were divided on several parts.
1. Build calibration curves to be able to estimate the concentration of periplasmic DNA using transformation
2. Figure out confidently obtained the right amount of periplasmic DNA (~9 ng for long fragments and ~ 2 ng for short)
3. Figure out the degree of chromosomal contamination during the extraction by using an uptake essay of donor DNA and recipient cells marked with different antibiotic genes
The first goal was with success (well almost), I was able to build a calibration curve using Map7 genomic DNA with a level of detection between 0.09 and 90 ng of DNA. A small problem here was that I used by mistake non-sheared Map7 DNA instead of the 6kb sheared DNA; but given previous results it is safe to assume that this curve would be useful to give me a first approximate estimation. I will validate this by building a calibration curve with 6kb sheared DNA.
I made another calibration curve using 250bp average sheared genomic fragments of Map7. This curve also looked very good. However, given the fact that small fragments have less uptake given that some of them lack a USS site, the detection limit was higher than with large fragments ( between 9 and 1000 ng) and it is not useful for my purposes (my expected yield is around 2 ng).
Given my results on my calibration curves, the second goal was achieved partially, I was able to estimate an overall yield of 9 ng for my 6kb fragments with almost no contamination from the washes (less than 0.09 ng). I was not able to estimate the yield on the periplasmic extraction using the 250bp fragments given the detection limits of my curve. I will use a fluorometric approach using pycogreen dye with help of another lab.
Third goal was not reached yet given many problems/mistake that happen along the experiments. For this experiment I first needed sheared DNA with a Nal resistance. I already had this sheared DNA, but since I did not prepared it, I needed to make sure that it had the right antibiotic resistance (Nal). I transform Kw20 cells with this DNA (NalR) but i did not had any colonies. Later, I realized that the problem might be some really old plates (6 months old) that I was using, since when dilutions were re-plated in fresh plates I saw ~1000 colonies in a 1e-2 dilution plate (I also run several controls of strains sensitive and resistant to Nal). I reconfirm this by repeating the transformation using fresh plates and I got 2.9e+4 transformants/ml (using 300 ng DNA). This results also suggested that I really need to make a calibration curve with the 6kb sheared DNA to validate my previous results.
Then I needed to generate competent cells from a strain with nov resistance (RR3129). First, when I incubate my cells in sBHI overnight I saw that my negative control was contaminated, so I could not start my experiment with a lot of confidence that my cultures were not contaminated too. I started repeated the overnight culture and the next day I made some competent cells. To my surprise when I tested this competent cells I did not get any colonies as well. So, I decided to repeat the transformation but this time transforming some kw20 (that I knew were competent) in parallel. The kw20 cells had expected level of transformation but the RR3129 cells did not had any colonies at all. This results show me that I probably made some mistakes during the process of making RR3129 competence cells.
Fortunately I able to identify the source of the problems by running good controls, but I need to include even more controls more efficiently. For example, I will repeat the procedure of making my RR3129 cells competent, but I will make some Kw20 cells competent as well. I will also test my plates by streaking strains sensitive and resistant to antibiotics after making them, and I will include this controls always after using somebody else plates (especially if they are not fresh)