How to get bands for plasmid extraction?

If you are not seeing any bands except for the ladder in your gel electrophoresis after plasmid extraction, here are a few things to consider and troubleshoot for your next attempt:

Concentration and Amount of DNA: Ensure that you are using an appropriate volume and concentration of plasmid DNA in the gel-loading buffer. If the concentration is too low, the bands may be faint or not visible at all. Quantify the DNA using a spectrophotometer or a fluorometer to ensure that you have an adequate amount of DNA for visualization.

Gel Electrophoresis Conditions: Verify that the gel electrophoresis conditions are set correctly. Ensure that the gel percentage, running buffer, and voltage applied are appropriate for the size range of your expected plasmid bands. Lower voltage and longer run times can help visualize smaller plasmid DNA, while higher voltage and shorter run times are suitable for larger plasmids.

DNA Integrity: Ensure that the plasmid DNA is intact and not degraded. Plasmid DNA can be susceptible to degradation by nucleases or physical shearing. Handle the DNA samples gently and avoid excessive heat or mechanical stress. Verify the quality of the plasmid DNA by running a positive control (known plasmid DNA) alongside your samples.

Loading Buffer: Check the composition of the loading buffer used. Make sure it contains a suitable tracking dye, such as bromophenol blue or xylene cyanol, to monitor the migration of DNA during electrophoresis. The loading buffer should also have a high enough density to allow the samples to sink into the wells properly.

Loading Volume: Ensure that you are loading an adequate volume of DNA into the gel wells. Insufficient volume or uneven loading can lead to poor band visualization. Make sure to pipette the samples into the wells slowly and evenly.

Staining and Visualization: After gel electrophoresis, use an appropriate DNA stain, such as ethidium bromide or SYBR Safe, to visualize the DNA bands. Follow the staining protocol carefully, including the incubation time and concentration of the staining solution. Ensure that the gel is adequately exposed to UV light to detect the stained DNA bands.

Check Equipment: Verify that your gel electrophoresis equipment, including the power supply and UV transilluminator, is functioning properly. Check for any potential issues with the equipment that could affect the visualization of the DNA bands.

By considering these factors and optimizing your experimental conditions, you should be able to obtain visible bands for your plasmid extraction in the gel electrophoresis.

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Robert Adolf Brinzer

Have you checked the concentration of your extracted DNA? You need >50ng for a band to be visible.

Hong Thai Le Nguyen

I don't know what protocol you use during extraction, so there are many points that can affect the results.

- What is the OD that you use when extracting?

- Is there a step to check the infection process?

- The plasmid size and their concentration (how many ul elute buffer do you elute)?

Shehreen Sohail

No, I didn't know about OD and I don't know how to find it.

I don't know about the strain you use. But I use plasmid extraction from proliferative e.coli strains (fast proliferative vector- E.Coli TOP10) and usually grow to an OD of about 0.4 (600nm wavelength). (It is used to determine the concentration of microorganisms in the sample)

Anirban Mukherjee

check concentrarion of eluted plasmid before running a gel... probably use a nanodrop if available..

also try going with lower amounts (40-50ul .. usually works for me in case of ecoli strains) of elute buffer to get higher conc of eluted DNA .

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