Some Gram positives you should include: Staph aureus, Streptococcus pyogenes, Bacillus subtilis, Enterococcus faecalis. Then I would also include a Mycobacterium, as they are really though, if you can't use M. tuberculosis for safety issues, you can try M. smegmatis. I don't know much about difficult to lyse gram negative species, but I think you should at least include E. coli as a positive control.

Many thanks Francesco.

You clearly want to have several Gram+ cells as suggested by Francesco, but I would go further in his suggestions and add several Actinobacteria if you can. Their cell walls are difficult to break open, so it would be interesting for you. Also, Archaea cells would be very interesting to look into, but I'm unsure how many are readily available in culture, and how relevant they would be to your system. And of course, always have a positive control to compare the effectiveness of the different extractions to.

Many thanks, Corinne and Francesco.

We've compiled the list below as a starting point, to try and capture some important test parameter,. to test dna extraction protocols. With the aim of determining a short list of 'good' generic methods, amenable for NGS automation. Hence some of the parameter headings such as phages/plasminds (ie. contains phages and/or plasmids).

If there are any other test species you think would be good to add, please let me know. Also, we'll try and look at some Archea but it may be a little trickier...

the list:

1 Gram positive cell wall Streptococcus pyogenes

Staphylococcus aureus

Mycobacterium avium

Streptococcus pneumoniae

 M. smegmatis

2 Gram negative Haemophilus influenzae

Escherichia coli

Campylobacter

3 Acid-fast Mycobacterium fortuitum

Nocardia

(M. avium)

4 plasmids* Enterococcus faecalis

Clostridium difficile

Acinetobacter baumannii

Klebsiella pneumoniae

Neisseria meningitidis

(Staphylococcus aureus)

Pseudomonas aeroginosa

(Staphylococcus aureus)

Salmonella typhimurium

E coli

5 Phages* (Salmonella typhimurium)

(Acinetobacter baumannii)

(Escherichia coli)

6 large genome (Pseudomonas aeroginosa)

Achromobacter xylosoxidans

Amycolatopsis mediterranai

7 small genome Haemophilus influenzae

Aquifex aeolicus

(Streptococcus pyogenes)

8 high GC (Pseudomonas aeruginosa)

Corynebacterium diptheriae

9 low GC (Clostridium difficile)

Fusobacterium nucleatum

10 Different growth conditions (Clostridium difficile)

Mycobacterium avium

Bacteroides fragilis

Micrococcus

11 spore-formers Clostridium sporogenes

Bacillus subtilis

That seems like a very good list to start with!

Good luck!

Hi!

I would add Rhodococci as well as to this excellent list. I had really hard time with them, but I came up with a good solution just by adding 600 mg/l ampicillin for 4 h, prior to DNA extraction that weakened the cell wall greatly.

Good luck,

Istvan

Thats good to know, I had thought of this bacterium...