Has anyone ever tried something new during the plasmid isolation from bacteria cells which resulted in improved plasmid yield?
Suggestions can cover centrifugation, changing the concentration of all 5 solutions, storage or anything. Please specify the change and increase in yield which observed?
Regards
Jitendra
Hi Jitendra,
Are you isolating plasmids from E. coli or plasmids (binary vectors) from Agrobacteria? Generally, binary vectors are low copy number and you will obtain much less plasmids from Agrobacteria than from E. coli when you do miniprep. By the way, are you using a commercial kit for isolating your plasmids? I use kit for isolation, at the final elution step by adding TE or water to the tube, I let it stands for a bit longer time than the instruction suggests before performing centrifugation to collect the eluent.
I found that when doing spin column extraction and purification of plasmid DNA, using pre-warmed nuclease free water and a slightly extended incubation period and centrifugation for the final elution does give you slightly better yields.
I find the Roche kit very good. You get high yield if you have multicopy plasmids.
Hi Jitendra
You can try this. First you need three solution: solution 1 (50 mM glucose; 25 mM Tris-HCl ph 8.0; 10 mM EDTA 8.0) solution 2 (0.2 N NaOH, 10% SDS) Solution 3 (5 M CH3CO2K, 11.5 % acetic acid glacial). Solution 2 is prepared at moment.
For 5-10 ml of overnight bacterial culture cell use this volume
all in ice
1. Pellet resuspen in 200 ul of solution 1, add 200 ul solution 2 at temperature room. Incubate for 5 min on ice.
2. Add 200 ul solution 3, mix gently and the sample centrifuged at 13,000 rpm/5min/4°C.
3. The supernadant was mix with 600 ul phenol:chloroform:isoamilic alcohol.
4. Centrifuged at 13,000 rpm/4min/4°C.
5. Take the organic phase (superior phase) and add 600 ul cold ethanol absolute, and centifugated for 8 min at 4°C
6 Washe the pellet with ethanol 70% and finilly resuspend the pellet on 30 ul of H2O ultra pure.
This method is great to increase yield plasmid. I hope it is usefull
Dear Kashmeel Maharaj!
Thanks for your suggestions. they were were interesting.
Dear Jose,
Thanks for your suggestion . it was indeed helpful!!
Dear Yuan-Yeu Yau, and Jana Schmitzova,As you asked, I am not using any commercial kit. Thanks for your suggestions regarding usage of kit!
I follow a similar protocol than jose. But, in instead of add phenol, which is something bothering and toxic, after step 2 I take the supernatant and I add 0.5 vol of isopropanol or 2 vol of ethanol and storage at -20C for about 20min so that most of plasmid DNA can precipitate, then centrifuge at 14k rpm for 25 min. you will have a white pellet which you can continue with the step of the ethanol 70%, centrifuge and resuspending in water or TE buffer.
Thanks for your answer Marcos. I agree that phenol is a concern during plasmid isolation.
Thanks for your suggestions. It was indeed helpul.
regards
Jitendra
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