Hi everyone,
I've been trying very unsuccessfully to transform a lux plasmid into a staph strain.
I'm trying to go the transduction route now instead.
What I am wondering is how to go about it? Does anyone have a staph specific protocol or failing that a gram positive protocol or maybe an informative link??
Thanks alot!
Jen
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#2
phage434
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Posted 08 July 2009 - 05:24 AM
Can your plasmid replicate in Staph? Few gram - plasmids will function there.
#3
jen_23
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Posted 08 July 2009 - 10:13 PM
phage434, on Jul 8 2009, 02:24 PM, said:
Can your plasmid replicate in Staph? Few gram - plasmids will function there.
The plasmid was specifically made for staph strains. my prob is that I'm trying to transform clinical isolates and it's just not working out. I've been trying for several months with several different protocols and getting no where ????
#4
phage434
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Posted 09 July 2009 - 04:26 PM
There can be problems with restriction systems. Staph has a Sau3aI system cutting at GATC, protected by methylating the C. I don't believe the methylase is available commercially, unfortunately. You could PCR your plasmid using 5-methyl cytosine dCTP (which is commercially available) to protect it, if this is the problem. I believe some plasmids transform depending on your luck, but with very low efficiency. You could also mutate out the GATC sites, depending on how many there are. If you need a tetM gene without GATC sites, I have one in the freezer (for exactly these reasons). What plasmid are you transforming?
