As I described in my previous posts, my objective in the following weeks is to extract DNA from the periplasm of competent Rec2 cells and be able to effectively quantify the DNA, assess the quality of the DNA and assess the amount of chromosomal contamination in the extract.
To assess this I did an uptake experiment using competent Rec 2 cells and incubating them with 100 ng of a PCR product of 6kb size from the GYRB gene. Then, I extracted the periplasmic DNA and I run the entire extract in a 1% gel with a 1kb GeneRuler ladder (NEB).
Results were disappointing, I saw a lot of RNA contamination, a very faint band of chromosomal contamination and a almost non visible band of my periplasmic extract band. I made a judgement mistake in believing that I could quantify the band only using a ladder, so I repeated the experiment (this time using 3 replicates) and loading the periplasmic extract at the same time as the input PCR product + 100 ng of MAP7 DNA + 10 ng of MAP7 DNA + 1ng of MAP7 DNA +3ng of MAP7 DNA. Unfortunately, during the periplasmic extraction the stupid centrifuge broke the epperdorf tubes of two of my replicates. I tried to same the last replicate however the extraction did not worked very well as I was only able RNA contaminant. Additionally the dilution of the MAP7 DNA in the gel did not worked as planned, I was able to see clearly the 100 ng band, but the 10 ng band was very faint (the rest of bands were not visible).
From this point I decided to stop and plan my experiment more carefully, as well re-think the strategy.
Lets examine what I know so far:
- During last experiments I was able to determine that adding 1ug of sheared 6kb 86-028NP DNA I am able to recover from 4 to 6 ng of periplasmic DNA. But why this amount of DNA was not visible in the gel?, well…. the 10 ng band of MAP7 was barely visible too.
- In another experiment I determined around 4 ng of chromosomal contamination (however I need to confirm this estimation). The faint band visible in the gel make me believe that I have 10 or less nanograms of chromosomal contamination.
- The gel at least showed me that the chromosomal band is intact. This will allow me to purify periplasmic DNA from chromosomal DNA using a size exclusion method.
- Previous uptake experiments using radioactive labelled sheared DNA indicate that Rec2 competent cells took up up to 9 ng of 6kb sheared DNA (when 400 ng of DNA were added to 0.5 ml of cells). This suggest that probably I am loosing up to 40% of the periplasmic DNA during the organic extraction steps. However, the only way to confirm this will be to make a uptake experiment and organic extraction with radioactively labelled DNA.
- I still have no idea of how much periplasmic DNA I am recovering from uptake experiments using the 0.25kb sheared fragments. Considering that radioactively labelled experiments told me that competent cells are taking up around 1 ng of 0.25kb DNA, I am likely recovering around 0.5 to 0.6 ng of periplasmic DNA.
Considering that the minimum amount of DNA needed to make sequencing libraries using the Nextera XT system is around 1ng, I am confident that I am recovering enough periplasmic DNA from the 6kb fragments. The problem is that I have no idea of how much degradation is happening in those fragments. It seems that ethidium bromide does not have enough resolution to detect confidently low amount of DNA, so how can I assess the quality of the periplasmic extract? The only way to assess quality of DNA seems to be to use radioactively labelled periplasmic DNA and run that DNA in a gel. The gel could be dried and reveled using a phosphor screen. However, I am not very worried about degradation since Barouki & Smith (1985) showed that periplasmic DNA in rec2 cells is still intact after 60 minutes of incubation.
This figure from the paper shows radioactively labelled linear plasmid DNA (pCML6) and chromosomal DNA (Chr) extracted using the organic periplasm extraction procedure in wild type, Rec1 and Rec2 mutants. In wildtype and Rec1 most of the periplasmic DNA is either degraded or recombined after 30 minutes of incubation while in Rec2 half of the DNA is still intact after 60 minutes of incubation.
I am more worried about increasing the amounts of periplasmic DNA for 0.25kb fragments. This might be a problem since assuming that am recovering around 0.5 ng per 1ml of competent cells, I would need pool at least 4 samples to have 2 ng of DNA. however this DNA is not purified yet, and it still has several amounts of RNA contamination. I can eliminate RNA contamination by adding a RNase A step just after recovering the aqueous phase of the chloroform extraction step. But this step also requires an additional phenol:chloroform step. Each additional step decreases the amount of periplasmic DNA recovered and additional purification by size exclusion would decrease the amount of periplasmic DNA even more.
What to do next?
Right now the best thing to do is to confirm my previous results by measuring the amount of periplasmic DNA and chromosomal DNA with biological replicates. This is done as described previously in my February 04 post. In this new replicates I can introduce a RNase A step.
Once I finish this I can use a size exclusion technique to purify the periplasmic DNA and then quantify the DNA using pycogreen. Pycogreen allow me to detect up to 50pg of DNA and since sample would already be purified then the yield estimation would be really accurate. This quantification would allow me to detect periplasmic DNA of 0.25kb sheared fragment extractions.
I would still need to increase the amount of periplasmic DNA obtained. This could be solved by making an mega-uptake reaction using 5 ml of competent Rec 2 cells with 1 ug of sheared NP DNA , then pellet all the cells in a single tube and extract the periplasmic DNA. I could even try extracting the DNA from several different amounts of Rec2 cells and the measure how that improve the amount of periplasmic DNA obtained.
I can incubate 1ml, 3ml,5ml,10ml of Rec2 competent cells with 1ug of DNA and then extract periplasmic DNA and quantify that DNA.
At the end of this I would still not know the quality of the DNA, but if the previous idea work and I can duplicate or triplicate the amount of periplasmic DNA by starting from 5 – 10 m of competent cells then I might also see the periplasmic band in gel without needing radioactive isotopes (at least for the 6kb fragments).