5 replies to this topic

[Nrelo]

After several times of failure in R.E digestion, I suspect that my plasmid is degraded by DNase since the host strain is not a endA- one. Can anyone suggest a good way to remove/inactivate DNase after mini-prep without affecting downstream cloning process (R.E digestion etc)? Thanks a lot.

[perneseblue]

Nrelo, on Mar 11 2009, 08:09 PM, said:

After several times of failure in R.E digestion, I suspect that my plasmid is degraded by DNase since the host strain is not a endA- one. Can anyone suggest a good way to remove/inactivate DNase after mini-prep without affecting downstream cloning process (R.E digestion etc)? Thanks a lot.

what kind of extraction method are you using?

In general to overcome this problem is to work fast during the extract protocol. keep the sample on ice. Use cooled reagents. Increasing the molarity of EDTA used would also help. These steps would slow down the plasmid degradation.

I would use phenol chloroform extraction to inactivate the DNAse.

[HomeBrew]

The easiest thong to do would be to retransform your intact plasmid into a endA- host, and work on it from there.  Will your plasmid replicate in E. coli?

[NemomeN]

Do your miniprep like normal and then incubate your prep at 65-70 degrees for 20 minutes and you're all set.

[Nrelo]

NemomeN, on Mar 12 2009, 06:12 AM, said:

Do your miniprep like normal and then incubate your prep at 65-70 degrees for 20 minutes and you're all set.

ya i do miniprep as usual. i will try you method tommorrow. It seems that it is much more easier. Thanks a lot

[Nrelo]

HomeBrew, on Mar 12 2009, 04:49 AM, said:

The easiest thong to do would be to retransform your intact plasmid into a endA- host, and work on it from there.  Will your plasmid replicate in E. coli?

my plasmid contain R6K replicon which only replicate in e.coli lambda pir strains (e.g S17 lambda pir and CC118 lambda pir), unfortunately, both strains are endA+. I really wish I had a lambda pir endA- host