Hello Everyone! I use Golden Gate technology to build plasmids in my lab. I have a plasmid that I've built that I'm now trying to maxi prep. I've already done a miniprep on this plasmid and sent it for sequencing and it was verified to be what I expected it to be. So I took this DNA and transformed it again to do a maxi prep. For both the Mini and Maxi Prep I used NEB5a cells. I've done the maxi prep on this plasmid twice and each time I'm left with just the antibiotic resistance and promoter. I have no idea where the rest of my insert is going. The GOI, Terminator, introns etc are all disappearing.
The source DNA that I'm using is sequencing correctly. I have no Idea what is happening during the maxi prep process.
It sounds like you're experiencing some frustrating results with your maxi prep process. I am no expert in the process, however, I found your question quite interesting so I did some research. Here are a few things you could do to troubleshoot and try to fix the problem. I hope it helps.
1. Check Plasmid Integrity: After maxi prepping, run a gel electrophoresis of your purified DNA to check for the presence of the insert. This will help you determine if the insert is being lost during the purification process or if it's not being maintained in the plasmid.
2. Optimize Maxi Prep Protocol: Ensure that you're following the maxi prep protocol correctly and that all steps, including cell lysis, DNA binding, washing, and elution, are performed properly. Consider using a different maxi prep kit or protocol to see if the issue persists.
3. Use a Different Host Strain: NEB5a cells are suitable for cloning but may not be ideal for maxi prep due to potential issues with plasmid stability or copy number. Try using a high-copy-number strain such as DH5α for the maxi prep to see if it improves the yield and stability of your plasmid.
4. Check for Nuclease Contamination: Ensure that all reagents used in the maxi prep are free from nucleases, as they can degrade the insert. Use fresh reagents and avoid contamination during the procedure.
5. Verify Transformation Efficiency: Confirm that your transformation efficiency is sufficient for the size of the plasmid and the complexity of the insert. Consider optimizing the transformation protocol or using a different competent cell preparation.
6. Consider Fragment Size and Stability: If your insert is particularly large or contains repetitive sequences, it may be prone to deletion or rearrangement during replication or purification. Try using smaller fragments or modifying the insert sequence to improve stability.
7. Perform Additional Sequencing: If possible, sequence the purified DNA after maxi prep to verify the presence of the insert. This will help you determine if the insert is being lost during the prep or if it's not maintained in the plasmid.
By troubleshooting these potential issues, you may be able to identify the cause of the disappearing insert and improve the success of your maxi prep process. Please let me know how you end up fixing the problem.
The first thing is to make sure this wasn't just a fluke. I would retransform again and screen a few colonies by mini prep to be sure they are correct then do a maxi prep from one of those confirmed cultures. It is not unknown to get plasmid deletions, especially if there is a strong selection against the insert (or expression of the insert).
Michael J. Benedik, I did a mini prep and confirmed it was correct through sequencing and then used that DNA to maxi prep. The mini Prep was fine but when I do a maxi prep the insert is gone. I'm not sure how this happens. I used the same cells to do both. This is the second time i've done a maxi prep on this plasmid from the same confirmed mini prep and still same results. The insert isnt there but the resistance gene is and the promoter is.
If you have a picture of the gel you ran to confirm the mini prep is correct that might be useful to look at and maybe see if there is something obvious.
Michael J. Benedik So I think what I'm going to do is re transform this and streak it on a plate. Pick colonies, do mini preps and sequence them. and then from the cultures I grew I'll use that as my starter culture to inoculate for Maxi Preps instead of just retransforming from the miniprep. When I miniprep that's coming from one single culture, but when I maxi prep its a mix I don't do single colonies I just take a loopful of the transformed bacteria. I guess theoretically it should work because there is antibiotics in the media that I'm inoculating for my starter culture and antibiotics in the media that I'm inoculating for my maxi prep, but anything that has the resistance gene will grow even if it has nothing else. So it could just be outcompeting the complete plasmid and I'm getting an abundance of the ones that don't have anything but the resistance genes
Michael J. Benedik This might be exactly what you just suggested lol now that I go back and reread your response
If you're observing a disappearance of a portion of your plasmid, there are several potential reasons for this phenomenon:
Did you eventually manage to get a good plasmid prep of your clone?
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