Dear collegue,

I worked with S. aureus PFGE using smaI restriction enzyme and the PFGEs gone well. What restriction endonuclease and Marker or reference standard did you use? I used S. aureus strains NCTC 8325 as reference standard. If you want I can send you the protocol I used (

sorry ...

the protocol I used (with the parameters for the PFGE run). I hope this could be useful for your work.

Bye

Vincenzo

Dear collegue,

I use the same NCTC as reference standard and smaI as RE. I will be grateful for your protocol, thanks in advance!

here you may see the run-protocol

-> (Volts/cm = 6, Included Angle= “+60/-60” Ramping Factor = A “Linear”)

-> Block 1(Blk1) State 1 (St01) [6.0] V/cm a= [linear]

Ang = [+ 60] InTm = [5 seconds] FnTm = [15 seconds]

-> Block 1(Blk1) State 2 (St02) [6.0] V/cm a= [linear]

Ang = [- 60] InTm = [5 seconds] FnTm = [15 seconds] Migration Time 8 Hours

-> Block 2 (Blk2) State 1 (St01) [6.0] V/cm a= [linear]

Ang = [+ 60] InTm = [15 seconds] FnTm = [60 seconds]

-> Block 2 (Blk1) State 2 (St02) [6.0] V/cm a= [linear]

Ang = [- 60] InTm = [15 seconds] FnTm = [60 seconds] Migration Time 11 Hours

As you can see in the attached image (sorry for the low resolution), I wrote my idea and analysis.

It is a run with two NCTC strains, i wrote where the two blocks acts, in fact they are usefull to separate different sizes of fragment:

1st block for 200 kb to less then 50 kb;

2nd block for bigger then 200 kb

The critics is in the middle of the gel (the other bands are comparable with literature).

Do you think it could make sense?

Thank you again

see in this article protocole with Beckman GenelineTM Transverse Alternating Field Electrophoresis (TAFE) system

Dear collegue,

I saw your image and I think is quite good. In some cases could be happen that the bands in the middle of the gel (262 Kb and 257 Kb for NCTC 8325) are very close. You can see the in link below the NCTC 8325 PFGE profile with the two bands very close.

Your protocol is very similar to mine, just few differences:

block 1: run time = 11 hours

block 2 state 1 and 2: final switch time 45 sec.

In attachment you can find a pdf file with some suggestion to solve different problems related with PFGE.

I hope that the information could be useful for your work.

Bye,

Vincenzo

This is the link!!!!!

http://microgen.ouhsc.edu/s_aureus/s_aureus_home.htm

Have you finally solved the problem? I have quite experience in PFGE of S. aureus. If you need some help just give me a touch. It would be very helpful if you upload the image of the gel

I tried changing the run parameter but nothing to do.. I'm working with the NCTC 8325 to harmonize my lab and the protocol seems to be really simple. I add two pics below! thank you

Here another image

MURCHAN, Stephen, et al. Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains. Journal of Clinical Microbiology, 2003, 41. Jg., Nr. 4, S. 1574-1585.

Finally, I restart my experiments using another isolate.

In a CHEF-MAPPER equipment, I used the following parameters: 15-15s / 8.5h (block 1), followed by 15-60s / 11.5h (block 2), 6V/cm, at 14°C without interruptions and included angle 120° (or multistate option: state1 (vector1) +60, state2 (vector2) -60 for any blk).

Good Day Dr Guerrino Macori, how was the results? have you overcome it?