I am unable to get purified plasmid after the gel purification of my PCR amplified product(2kb size). I tried both ways like kit method and manual like I prepared my own plasmid binding buffer, checked for both gel elution and direct pcr product? but no purified is thereafter checking on 1 % agarose gel
Sorry. Dont quite understand your question
Are you saying you cloned your purified PCR product into your plasmid but were then unable to recover the plasmid after purification
Can you be more specific?
Do you mean that when you analyse your elution on an agarose gel you don't see it ? If both your direct PCR products and your elution does not show any product I would assume that your PCR didn't work simply
Dear Kirankumar Nalla
I think you are facing some trouble in gel clean-up step. You could follow these suggestions:
- Do not incubate the gel with plasmid binding buffer more than 60 degree centigrade.
- Please check whether the spin column is dry properly after the washing step (because the wash buffer generally contains alcohol, which reduces the solubility of DNA/RNA in elution step, if present). You can follow a dry spin for one minute following the last washing step.
- Incubate the spin column with elution buffer little longer than usual time mentioned in the protocol for better binding.
I hope it would help you. PCR product size more than 10kb is usually difficult for gel extraction. I wish you success very soon.
If I understand correctly, you are amplifying your plasmid by PCR (like for a site directed mutagenesis or to amplify the backbone) and then you are isolating this product from the gel. So you see the product once the PCR is done, but after purification, it disappears. Is this correct?
Even if there are no technical errors (e.g. you forget to add ethanol to your wash buffer or something like that) gel purification is usually not very efficient. I suggest 1) double check your gel with the purified product - increase exposition to the max and see if there is a faint band that matches your product; 2) re-do your PCR with 2x or 3x the volume so you start with more material in the first place. 3) when using the kit, vortex your mixture when you are liberating the DNA from the gel; bind the DNA twice on the column in the subsequent step, 4) not sure what kind of alternative method you are using for gel purification, we had some kind of mesh that we would filter the gel slice through and this method was what gave me best yields (you purify the DNA with phenol/chl extraction afterwards and precipitate with etoh).
Good luck!
Ana Crnkovic - yes madam after pcr I am able to see amplification after running on 1% agarose gel, but surprisingly after gel purification, no plasmid in gel elution
Satyendra Mondal thanks you for your suggestions I will try with your steps for gel purification of my pcr amplified product
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