Hello, I have plasmid extraction problem, what is going wrong in your opinion?

So, it sounds like you have your vector, did a successful transformation (obtained colonies), tested colonies by PCR, then didn't get any plasmid after the extraction.

One easy way to "lose" a plasmid is to not add antibiotic to the liquid medium during culture growth. Try again and double check that you are adding antibiotic to the broth right before you use it (don't store culture broth with antibiotics at room temp, the antibiotic breaks down).

Is anyone else having plasmid extraction issues? If so, it may be the kit. They do expire after about 1 year.

Hope this helps!

Waheed Abdullah

Sepideh Moori , could you please clarify that you don't see the band after digesting the plasmid with enzymes and see the band on DNA electrophoresis gel or after transfecting to the cells and observe on Western blotting. if it is the later case then it should be the reason I explained below. I wanted to construct one plasmid but it failed every time. Even the enzyme digestion test and sequencing results were right, but when I extract the plasmid, transfect into cells and observe it on the Western blot, I don't see any band. I was trying to express one gene from Hamster sequence, but it failed after many attempts, then I designed the primers from Human, cattle and pig mRNA CDS and used the mRNA source from the the above mentioned species cells and successfully constructed and expressed the plasmid. The actual problem is the CDS sequence sometimes the sequence available on NCBI is not verified by someone and the predicted sequence may have some issue. so my suggestion is to use other species sequence and I believe you will be successful. Good luck.

Sepideh Moori

Amy Klocko, thank you so much. I'll check the antibiotic and extraction kit.

Nikolay Klyashtornyy

If you are not observing any visible bands after plasmid extraction, there could be several potential issues that may have led to this problem. Here are some possibilities to consider:

Low plasmid yield: It's possible that the plasmid yield from your extraction was too low to detect on the gel. This can occur due to factors such as low bacterial density in the culture, incomplete lysis of the bacterial cells, or inefficient DNA binding to the extraction matrix. Consider optimizing the plasmid extraction protocol to improve the yield, such as adjusting the culture conditions, increasing the cell density, or using a different extraction kit.

Inefficient cell lysis: Incomplete lysis of bacterial cells can result in insufficient release of the plasmid DNA. Ensure that you are using an effective lysis method and appropriate lysis buffer to completely disrupt the cells and release the DNA. Consider optimizing the lysis conditions, such as extending the incubation time or increasing the lysis buffer volume.

Contamination: It's possible that your plasmid extraction has been contaminated with other substances that interfere with the DNA visualization on the gel. Contaminants such as proteins, RNA, or residual reagents from the extraction process can inhibit DNA migration or obscure the bands. Ensure that you follow proper purification protocols and perform appropriate purification steps, such as ethanol precipitation or column purification, to remove contaminants.

PCR-related issues: If you successfully observed the PCR band from the colony, the absence of the plasmid band after extraction suggests a problem with the extraction process rather than the PCR. However, it's important to confirm that your PCR conditions are optimized and specific for the target gene. Check the primer sequences, annealing temperature, and amplification cycles to ensure they are suitable for your gene of interest.

Gel-related issues: Consider potential issues with the gel electrophoresis process itself. Ensure that the gel was prepared correctly and that the gel concentration is appropriate for the size of your plasmid. Check that the DNA loading dye was added properly to your extracted samples, and verify the voltage, run time, and buffer conditions used during electrophoresis.

DNA degradation: DNA can be degraded by nucleases present in the sample or by improper handling/storage of the extracted DNA. Ensure that you are working in a nuclease-free environment, use RNase-free reagents, and handle the DNA gently. Store the extracted DNA properly at the appropriate temperature to prevent degradation.

Technical errors: Double-check your plasmid extraction steps and make sure you are following the protocol accurately. Small technical errors, such as incorrect volumes, incorrect centrifugation speed or time, or missing steps, can affect the extraction efficiency.

To troubleshoot the issue, you can try repeating the plasmid extraction process while paying close attention to the steps mentioned above. It may also be helpful to consult with experienced researchers or colleagues who are familiar with plasmid extraction to get their insights and suggestions specific to your experimental setup.

These video playlists might be helpful to you:

https://www.youtube.com/playlist?list=PLzBiAPKi8vOPbE7Db9mKwloe34E8rXXEU

https://www.youtube.com/playlist?list=PLzBiAPKi8vONSqjcweFWHMRy13y3484bE

https://www.youtube.com/playlist?list=PLzBiAPKi8vOMheKwb0mu1dcZRaeGUFSlw

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