I want to do the plasmid midi research by 25ml DH5α-plasmid, but the concentration i got is very low, about 10 ng/ul. I followed the protocol every step, and i don't know the reason. Can i enrich the plasmid concentration by mini kit?
Can someone give me advice?
Low plasmid yields are usually caused by one of the following reasons:
1) low copy number plasmid. Check the origin of replication of your plasmid to find out information on copy number. If it is low copy number, use a low salt LB formulation, and you can also try chloramphenicol amplification protocols to boost yield.
2) selection pressure that causes your bacteria to lose the plasmid. If your antibiotic is bad, the selective pressure will be lost and plasmid-negative bacteria can overgrow your culture. Also, if your plasmid harbors a toxic gene product or repetitive sequences that make replication difficult, you can run into recovery issues.
3) poor lysis of your bacteria, either because you are using too much bacteria or your lysis buffer is old. This is easy to check- your solution should clarify completely when you add the P2 lysis buffer. If it remains opaque, your lysis step is not working properly.
Please include some more details about your plasmid (backbone, insert, total size), as this will help in the troubleshooting.
Low plasmid yields are usually caused by one of the following reasons:
1) low copy number plasmid. Check the origin of replication of your plasmid to find out information on copy number. If it is low copy number, use a low salt LB formulation, and you can also try chloramphenicol amplification protocols to boost yield.
2) selection pressure that causes your bacteria to lose the plasmid. If your antibiotic is bad, the selective pressure will be lost and plasmid-negative bacteria can overgrow your culture. Also, if your plasmid harbors a toxic gene product or repetitive sequences that make replication difficult, you can run into recovery issues.
3) poor lysis of your bacteria, either because you are using too much bacteria or your lysis buffer is old. This is easy to check- your solution should clarify completely when you add the P2 lysis buffer. If it remains opaque, your lysis step is not working properly.
Please include some more details about your plasmid (backbone, insert, total size), as this will help in the troubleshooting.
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