I performed plasmid isolation from Agrobacterium rhizogenes strain LB 510 and 072001, but no yield has been seen from electrophoresis. I'm using Fermentas Gene Jet Miniprep 50 and bacterial strain from my promotor.
Try heating your elution buffer to 70C and using 70 micro liters. This typically increases the yield of DNA. Also using heat shock instead of electroporation or other methods for transformations in the Agrobacterium genus increases the efficiency of transformation all together.
Increase you aliquote for high Plasmid extraction. Also increase your plasmid volume loading on gel..
I second Pawel Tulinski's comment, I've gone through thousands of miniprep columns from multiple manufacturers, and the columns go off. While it may be cost effective to buy a bulk kit, they go off relatively quickly and silently, and you're left with a bunch of useless columns.
Most lysis and neutralization buffers you can make at home too, if you are worried those are at fault.
Worst come to worst, you can use these buffers without a column to see if they are the problem. Simply follow the protocol with lysis and neutralization, pellet the precipitate, and add 2x volume isopropanol to precipitate DNA, spin for 15 min at max speed, and wash with 70% EtOH, resuspend in water or buffer.
Dear Yuda,
have you ever used plasmid isolation solutions for other plasmid isolation like from DH5 alpha? If you do not have any problem in your solutions, the problem should be in plasmid. please check your plasmid whether or nor plasmid is low copy or high copy. One another problem is plasmid containing suicide gene. good luck to you.
Oh..great Answer my friends..Thanks All..
@ Mr Ahmet Arman, no sir, my promotor didn't ask me to isolate plasmid from Dh5alpha. I will try to isolate plasmid again monday next week...
@ Mr Brian Lin thanks for suggestion...sir i would ask, are the protocol using PCI purification ? or modified from sambrook sir ?..before i think the sambrook protocol is more cheaper than using kit, but i still doubt to use it
@ Martha Giraldo, i don't use any appropriate antibiotic supplemented in LB medium, because my promotor just growing bacteria in LB before miniprep..i think this is one of the causative from my fault and i don't know what the plasmid type of the Agrobacterium strain LB 510 and 072001 strains, so i cannot to know what the antibiotic resistent character of it.
Maybe anyone knows about the type of the strain LB 510 and 072001 characteristic and the plasmid they contained ??? (i was searching but i still don't get it)
If your plasmid has a selectable marker for bacterial expression, such as amp, kan, etc resistance, you need to be using that when you isolate plasmid. Without antibiotic selection, the bacteria undergo no pressure to keep the plasmid, and you will get completely wildtype empty bacteria, both your strain which kicked out the plasmid, and also random bacteria that floated into your delicious LB.
Regarding the problem of not knowing the plasmid within your strain, does your supervisor know what plasmid they want in the first place? Someone must know either where they got the bacteria or what they want from it. Once you know what the plasmid is, or atleast the backbone, you will know which antibiotic to use.
This is actually a funny coincidence, because I just (today) had to help troubleshoot someone who was doing maxi-preps and didn't put antibiotic in the LB, and got nothing, repeatedly. Added the amp, and we were swimming in plasmid! I think once you find out which antibiotic to use, your original column minipreps will work fine. And if worst comes to worst, make 2 minipreps, one with amp and the other with kan, and see which one grows after you innoculate.
Good luck!
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