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Need a plasmid encoded with ampicillin resistance
Moderators: AmyCowen, kgudger, MadelineB, Moderators
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deleted-392869
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- Occupation: Student
Need a plasmid encoded with ampicillin resistance
I am a student working on a project with transformation, but have not taken any classes involving biology, so it has been difficult to teach myself microbiology. I know I need to use GFP tagging to locate which E. coli take up the ampicillin resistant DNA. I plan to use a restriction enzyme (not sure what type I would need) with an r7 promoter to use plasmids (not sure what type) to use the shock heat method. It would be very helpful if someone had a protocol explaining how to prepare DNA and E. coli for the transformation process. Most websites merely have prepared cells for transformation or do not have procedures with specific plasmids or enzymes to use. Thank you!
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EAMills
- Former Expert
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Re: Need a plasmid encoded with ampicillin resistance
Hello Dyanimarie,
I would recommend (if possible, since they are quite expensive) buying already prepared chemically competent cells (those are the type that you can transform using the heat shock method). There are protocols available for preparing one's own competent cells (I do this with some regularity), however it is difficult to do unless you can access to a biology laboratory, and you need some starter bacteria to get the process going (which is usually from a commercial stock). DH5alpha cells are a very useful strain (of E.coli cells), and generally the most inexpensive.
The reason that the protocols you see are all general is because the transformation procedure is dependent on the exact strain of cells you use. For instance usually you do a heat shock at 42 degrees C, but the exact time (usually around 30 seconds) depends on exactly what strain of cells you have.
In terms of restriction sites, it depends on exactly what plasmid you have. Many of the commercial kits of competent cells come with a control plasmid, although they are unlikely to have GFP in them, however they do have antibiotic resistance, so you could grow the transformed cells on a plate with antibiotic resistance (ie amp) and only those which have the plasmid will grow. Once you have a map of the plasmid you can determine which restriction sites to use to do a diagnostic digest. -
In terms of purifying the plasmid from the E.coli, you do plasmid purification procedure called a miniprep. There are also commercial kits for this (but again are expensive), another method is a standard alakaline lysis prep, however this also involves reagents you would find in a molecular biology lab but not in your home.
I hope this provides a good starting point. Once you have a better idea of the specifics, we would be happy to help you figure out the details.
Elizabeth
I would recommend (if possible, since they are quite expensive) buying already prepared chemically competent cells (those are the type that you can transform using the heat shock method). There are protocols available for preparing one's own competent cells (I do this with some regularity), however it is difficult to do unless you can access to a biology laboratory, and you need some starter bacteria to get the process going (which is usually from a commercial stock). DH5alpha cells are a very useful strain (of E.coli cells), and generally the most inexpensive.
The reason that the protocols you see are all general is because the transformation procedure is dependent on the exact strain of cells you use. For instance usually you do a heat shock at 42 degrees C, but the exact time (usually around 30 seconds) depends on exactly what strain of cells you have.
In terms of restriction sites, it depends on exactly what plasmid you have. Many of the commercial kits of competent cells come with a control plasmid, although they are unlikely to have GFP in them, however they do have antibiotic resistance, so you could grow the transformed cells on a plate with antibiotic resistance (ie amp) and only those which have the plasmid will grow. Once you have a map of the plasmid you can determine which restriction sites to use to do a diagnostic digest. -
In terms of purifying the plasmid from the E.coli, you do plasmid purification procedure called a miniprep. There are also commercial kits for this (but again are expensive), another method is a standard alakaline lysis prep, however this also involves reagents you would find in a molecular biology lab but not in your home.
I hope this provides a good starting point. Once you have a better idea of the specifics, we would be happy to help you figure out the details.
Elizabeth