We are working on vector isolation in the lab, the second time we did the isolation we got a concentration of 50ng/ul which is to lower then desired. The protocol followed siad to use a 1.5 ml eppendorf and add in total 6 ml of bacterial suspension and centrifuge it to create the pellet. We used 8 ml to create a bigger pellet, as well as using 2 eppendorfs side by side to have a greater total pellet and more DNA. The buffers were added but the concentrations were not changed, as the buffers were made for 6ml. But we think that 6 - 8 ml is not a big change and if anything the buffers would be saturated with DNA. The supernatant of both eppendorfs were then added to same spin column, and then after the centrifugation 50ul of TE buffer was added to elute the DNA. Other classmates got up yo 88ng/ul instead of our 50ng/ul and they did not do the extra pellet steps. The teacher hypothesized that maybe the spin column was saturated with DNA and that some how negatively affected the elution. Can anyone help with this problem? even tips for higher DNA isolation concentrations are welcome.
Thanks to all!
Yes, the columns can be oversaturated. I am not sure how much. But if you scale up your plasmid purification with two columns versus one column you will get more more DNA with both columns than with one column. One thing that makes a difference is to add the elution buffer to the column and then wait over 10 minutes before centrifuging the column to elute your DNA. You will get more plasmid if you wait longer rather than spin the column immediately. It pays off to be slow sometimes.
I have eluted 30 uL of 300 ng/uL plasmid from 20 mL of bacterial culture with one spin column. I think it comes to luck, though. Sometimes I do the same thing and get as much as you from the same plasmid.
plasmid extraction yield also depends on the copy number of the plasmid. for instance I usually achieve up to 600ng plasmids when I extract PET group plasmids. I centrifuged about 10 ml of bacteria, then I continue the process with a larger amount of buffers (about one and a half times of each buffer). one of the key points in plasmid extraction is that the pellet would be well lysed. one of the other key point steps is that after the cell derby centrifugation step (after adding the neutralizing buffer) the supernatant would be well clear and cloud-like turbidities will not observe in it. In this case, it is recommended to repeat the centrifugation step again by refrigerated centrifugation until the cloudy turbidity disappears. at the end it you should centrifuge the supernatant in two steps and let the spin column dried completely after the final washing step. then add the elution buffer, let it to absorb for at least 10 min then do the final centrifuge step.
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