I am using Qiagen Gel extraction kit for gel purification of linearized plasmid. The procedure is followed as per the kit. if i load 1ug DNA in agarose gel, i am getting 600ng DNA or less after elution. can any one suggest any alternate method of purification?
Thank you!
Hi there,
Gel extraction is difficult for large DNA fragment (like linearized plasmid). 60% yield is quite good actually.
Dear Sivasankar Panickan
A few tips that can improve your yield:
- Use a less amount of elution buffer/ H2O to elute the DNA.
- pre-warming the elution buffer to 50-60 degrees and increase the standing time before centrifugation after adding the elution buffer, to works well and will release more of the DNA from the membrane so resulting in a higher yield.
- and at last, if you see the low quality for your DNA, Nicking can occur if the gel is left under UV light for too long when you cut your bands.
GOOD LUCK
How much amount of elution volume did you elute with?
How long is your linearized plasmid?
Other students also showed the same yield of purification?
In order to elute as much DNA as possible, please check your samples in gel extraction as below.
- the volume of elution buffer is enough?
- how the buffer is applied to the column, in the center?
- the buffer’s incubation time on the column is enough?
- the color of your melted gel is yellow, which indicates that the pH is optimum to bind DNA on the column?
If your sample has just a linearized plasmid, not other fragments, other ways to purify your linearized plasmid is PCI DNA extraction or MiniPrep.
Hi there,
Gel extraction is difficult for large DNA fragment (like linearized plasmid). 60% yield is quite good actually.
- Badges
- Science topic