I am trying to work with a plasmid of about 8.5 kbp which contains the hph gene. I received the plasmid from add gene and it grew up nicely and gave me a good yield of plasmid (500 ng/uL) after a Qiagen mini spin prep kit. It arrived in XL1 blue cells. I sent it for sequencing and got good coverage (~75%) across the entire plasmid.
My goal is to run site directed mutagenesis (quick change protocol with complimentary primers). I think that I've got the PCR tuned in as I get bands at the size I am expecting. When I try to transform either the Dpn1 digest product or the template, I get colonies but they are very small. When I transfer the colonies to liquid culture, they grow but very slowly and seem to stop at low density. Plasmid purification gives me a low concentration (15-30 ng/ uL) and I don't see the plasmid when it is run out on a gel. Does anyone have any insights?
Some things I've tried playing with,
-varying the concentration of hygromycin B. I've been working at 50 ug/mL which is on the low end of what I see people using. Bumping it up to 100 ug/mL does seem to cut down the number of small colonies but doesn't eliminate them.
-cell strains, I've used NEB5a cells directly from NEB, as well as 10-beta and stbl3 cells prepared from a neighboring lab. Their post-doc confirms that the cells he produces are highly efficient. The paper that describes the generation of this plasmid uses NEB10-beta cells from NEB, I have some ordered.
Thanks in advance!
How much was the volume of your culture used for isolation and purification plasmid by using a plasmid mini prep kit? You need to check all the reagents used for plasmid mini prep.
Hi Alemu,
I just 5mL culture for plasmid purification. I've been using a new Qiagen kit and they seem to be good. We've run enough of them that we're about to move onto a second kit.
To increase the yield of your plasmid, you can increase the volume of your culture into 10 ml and incubate for 16-24 hours to get high copy number of your plasmid. You need to check weather your plasmid is a high copy or a low copy type.
Good luck!
The story continues with the troubleshooting.
Somehow I missed that hygromycin selection should be done in low salt LB. I've done plates and liquid cultures with varying salt concentration. The hygromycin I have been using kills un-transformed cells at any amount of salt. Successfully transformed cells seem to grow the same at any concentration of salt. The cells transformed with the Dpn1 digest grow the best in low salt and do progressively worse up to the standard LB at 10g/L NaCl.
Currently checking to see if the salt concentration effects plasmid yield of the successfully transformed cells.
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