My Plasmid is about 64 kb having a T7 Promoter which I need to amplify. Can I use this plasmid directly as a template in PCR reaction using T7 primer? Can this amplification is possible with only a single primer (T7 Primer)? or alternatively should I single digest the plasmid and then using this linearized plasmid as a template for PCR using T7 Primer? In case of digestion after PCR which primer is better for amplification: T7 Primer or primer having sticky end sequence?
Please give your suggestions.
PCR reactions always require two primers hybridizing to opposite strands of DNA and bracketing the fragment you want to amplify. With the choice of primers featuring a 5' tail with the appropriate restriction recognition site, it is possible to flank the amplified segment with restriction sites that can be further utilized for restriction cloning.
You can use the T7 forward primer but it depends on where your insert is that you are trying to amplify. If it is too far (more than 100bp from site) than it will not work. Try finding a primer sequence that flanks both sides of your insert. 2 primers are always needed for amplification. Generally I use my restriction enzyme sites for primers. Addgene has a really nice explanation of how to design such if you look up "Restriction Enzyme Cloning".
can you provide details of the plasmid? particularly name of the plasmid and from where it was purchased and the insert length?
It is sometimes possible to amplify a PCR product directly from a plasmid. But if it doesn't work the first time, you will need to linearize the plasmid first. Use a rare cutter that will cut your plasmid only once and NOT in the region you want to amplify.
And yes, you have to have 2 primers for PCR.
But you can sequence from a plasmid template. Linear is best so you don't get as much secondary structure. There are lots of companies that will do the Sanger from your plasmid using the T7 promotor primers at no cost.
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