How may I pool DNA from miniprep?

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Michael J. Benedik

For your first questions, most people just grow their cultures all day or overnight as convenient and don't worry too much about the actual OD before pelleting to do a mini prep. It's just easier to grow overnight. But I would say at least above OD600 of 1.

For your second question, yes you can pool the elution products. But note this should ideally be done from the same culture (grow a 10 ml culture for example and do 3-5 mini preps from it, or grow multiple cultures from the same starting culture). You should not pick different colonies, do minipreps of each and then pool.

However I would NOT run a second column on the pooled plasmid, no need to do so. You can either just pool the eluants together, or if you are seeking higher DNA concentration then do an ethanol precipitation and resuspend in a smaller volume.

Mathangi Palanivel

Michael J. Benedik Oh ok thank you so much for the insights! I will follow these.

Ahmad Al Khraisat

The ideal OD600 before you can pellet your cells will depend on several factors, including the type of bacterial strain, the growth medium, and the specific plasmid you are expressing. However, as a general guide, an OD600 between 0.6 and 1.2 is usually considered appropriate for most bacterial cultures. It is important to note that over-culturing the bacteria can result in plasmid instability and decreased yield, so it is recommended to harvest the bacteria as soon as they reach the desired OD600.

Pooling eluted DNA samples can increase the overall concentration of your DNA sample, but you should exercise caution when doing so as the yield and quality of each individual sample may vary. Before pooling the samples, it is recommended to quantify the concentration of each sample to ensure that they are of similar quality. After pooling the samples, you can spin them again with a spin column to remove any impurities or contaminants and then elute the purified DNA again with elution buffer. However, you should keep in mind that the elution buffer can have an impact on the quality of the DNA, so it is important to follow the manufacturer's instructions carefully.

Ahmad Al Khraisat ok thank you so much!!

Hello!

The ideal OD600 before pelleting your cells will depend on the growth conditions you are using and the plasmid you are extracting. Generally, an OD600 of 0.5-1.0 is a good range for most E. coli strains and plasmids. However, it is important to keep in mind that a higher OD600 may lead to more bacterial cells and therefore more plasmid DNA, while a lower OD600 may result in lower yields. It is also important to ensure that the culture is in the exponential phase of growth, as this is when plasmid replication is most active.

If you feel that the concentration of your eluted DNA is too low, it is possible to pool multiple elutions together to increase the total yield. However, it is important to ensure that the total volume of the pooled elutions does not exceed the maximum volume that can be loaded onto a single spin column. Additionally, it may be necessary to concentrate the pooled elution before loading it onto the spin column to ensure that the final concentration is appropriate. To do this, you can use a concentration method such as ethanol precipitation or a DNA concentration kit before proceeding with the spin column purification. It is important to follow the manufacturer's guidelines for the concentration method you choose, as different methods may have different requirements and limitations.

I hope this helps, and good luck with your experiment!

Ahmad Al Khraisat Thank you! For number 2, may I check if you have any good protocol for ethanol precipitation of DNA before concentrating it further? I currently use the Iqagen miniprep spin kit to get a volume of 50uL of DNA in elution buffer. I am a bit confused as to the next steps that I should take to pool two elution products. Thank you!