We are working with a couple of plasmids which are rather difficult to isolate in satisfactory quantities. We tried chloramphenicol amplification on one of them and this made a huge difference and solved our problem. Unfortunately, the other plasmid codes for chloramphenicol resistance, so chloramphenicol amplification seems to be out of the question. Are there any alternatives to chloramphenicol for plasmid amplification?
Traditionally spectinomycin is used to amplify plasmids when you can not use chloramphenicol. However be aware that chloramphenicol or spectinomycin amplification does not work for all plasmids. As Alejandro points out it only works for plasmids using RNA's to initiate plasmid replication (like pMB1/ColE1 plasmids). Most plasmids with other origins of replication can not be amplified in this way because they rely on protein synthesis for replication.
Short answer: I don't know, so take this as wild speculation. Cm amplification is supposed to work by inhibiting protein synthesis for plasmids whose replicons (such as ColE1) rely mostly on short RNAs for regulating their replication. Therefore, perhaps using another protein synthesis-inhibiting bacteriostatic antibiotic might do the trick? Tetracycline, for instance, would fit that bill...
Traditionally spectinomycin is used to amplify plasmids when you can not use chloramphenicol. However be aware that chloramphenicol or spectinomycin amplification does not work for all plasmids. As Alejandro points out it only works for plasmids using RNA's to initiate plasmid replication (like pMB1/ColE1 plasmids). Most plasmids with other origins of replication can not be amplified in this way because they rely on protein synthesis for replication.
We have several plasmids that have Kan and Cm in the plasmid backbone. They have the puc Ori. Using Kan for selection works well.
*You can also use the sacB and sucrose media for selection
**Also you can lower the Cm concentration in the media....(most of BAC clones use Cm for selection and Cm concentration used is 12.5ug/ml instead of 25ug/ml)
***Using TB (terrific broth) media instead of LB may also help
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