During my recent plasmid extraction using the Yektatajhiz-plasmid DNA extraction maxi kit , I encountered a significant issue. The extraction yield was unexpectedly low, even lower than that of the miniprep kit. Additionally, the A260/280 ratio is about 1.4, indicating a high level of protein contamination. Could you help me understand the reasons for the low yields and provide suggestions for improving them?
Farnaz Pooresmaeil
Possible Reasons for Low Yield and Protein Contamination:
Suggestions for Improvement:
Implementing these steps should help improve your plasmid yield and reduce protein contamination.
Hi Kirankumar Nalla
how can overloading the column lead to low yield of DNA? If you load too much of DNA, it will pass through and not bind, because the column will be occupied by the DNA, right? So you should have high (saturated) yield of DNA?
Tomáš Hluska Overloading the column saturates the binding sites, causing excess DNA to pass through without binding. This leads to lower DNA yield because unbound DNA is lost in the flow-through. Additionally, overcrowding can hinder efficient elution, further reducing the yield.
Kirankumar Nalla yes, but what binds to the binding sites? Isn't it mostly plasmid DNA?
Kirankumar Nalla Thank you for your complete answer. I follow the kit protocol carefully for all the steps, but how can I be sure that the kit buffers are working correctly? Will only PH measurement be enough?
Also, I think bacterial culture quality and quantity are very critical; my kit only suggests using 120-240 ml bacterial culture medium but does not mention other details about the culturing process or even suitable OD; best on my searching OD between 2-5 could be ideal for plasmid extraction in large scale, Do you have any idea about this matter?
Farnaz Pooresmaeil
To ensure that the kit buffers are working correctly, pH measurement alone isn't sufficient. You should also verify that the buffers are fresh, properly stored, and not contaminated. Testing the buffers with a known positive control can also help confirm their efficacy.
Regarding bacterial culture, you're right that culture quality and quantity are critical. For large-scale plasmid extraction, an OD600 between 2-5 is typically ideal, as it indicates a dense culture that should yield a high amount of plasmid DNA. Ensure that the bacteria are in the late-log phase when harvesting for optimal results. you should monitor the growth curve of the culture by measuring the optical density at 600 nm (OD600) at regular intervals.
Kirankumar Nalla Thank you for taking the time to answer my question. I wish you luck.
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