I purified around 20 samples and failed to get enough concentration. When i extract the plasmid containing my gene from E.coli JM101, i get concentration from 100-250ng/ul. After that i use digestion enzyme and buffer to linzearize it (for transformation to p. pastoris), run the gel for confirmation and get correct band then try to purify it but unfortunately the DNA concentration either becomes low or disappear. I've tried thermoscientific (GeneJET Purification columns and collection tubes), SanPrep Column DNA Gel Extraction Kit, FastPure Plasmid Mini Kit. What could be the reason? any help
Note: I'm not purifying from gel
How many micrograms did you digest?
Just remember that when you are using any extraction/purification kit using columns you will lose up to to 60% of your DNA. So try to digest around 3ug of DNA. Then performing elution steps several times and incubating to increase DNA recovery.
Best
This should not be happening. If you're doing it right, it means some component is broken. Look at everything very carefully. Watch every drop of liquid. Does it go where you want it to go? Does anything at all look funny or not quite right? Is there any precipitate, any coloration or anything at all that you're not expecting?
Check the pH of everything. Lysis solution is strongly alkaline. Binding solution strongly acidic. Elution solution mildly alkaline. If the elution buffer is acidic, it will not work.
Troubleshoot at what step you're losing it. Does the DNA stay on the column, or does it never bind in the first place? Recover some of the flow through and run it on the gel, to learn where you're losing it.
That said, there are a couple of ways even a poorly working process can give you enough DNA:
- As others have said, use more starting material. Make as much of it as you easily can make. Load all of it through the column in multiple spins.
- At the DNA-binding step, recover the flow-through and load it again. 2x, up to 3x can increase your yield substantially.
- At the elution step, elute with buffer pre-warmed to 70C. Do this quickly, to prevent cooling minute volume back down during handling. Recover the eluate, and apply it a second time (elute twice). You can also add fresh elution buffer twice, if you don't mind the resulting larger elution volume.
Use little more DNA (~1-2 ug) to start-with and try electroelution.
Dominique Liger I'm using 100-200 ng/ul and its the concentration comes with me after plasmid extraction
Miguel Fernández-Niño Peramachi Palanivelu Thank you so much for your replies. I would try to use 1-3ug of DNA and would follow the steps as you mentioned. Hope it would help
John Schloendorn Thanks for your reply Sir. I've tried up-to 2x at DNA-binding step to recover as much DNA as i can and i have tried to take every steps carefully and though for the elution step i use ddH2O and every time before elution i pre-warm it at 60C.
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