Hello:
I was wondering if I would encounter problems with simply using PCR to amplify a fragment of a plasmid that is mixed with the raw genomic DNA from a bulk phenol:chloroform extraction of a plasmid rich e coli culture?
thank you! - Amy J.
Agree with Prof. Paul. Just make sure that your primers are specific to the plasmid.You may run BLASTn homology search of the primers against E coli genome to check if it binds to the bacterial genomic DNA. Rest you are expected to get very good amplification.
best of luck,
SD
You will get a good intensity product. There might be cross reactivity with virus sequence but the large amount of plasmid expected should give a strong amplimer
Agree with Prof. Paul. Just make sure that your primers are specific to the plasmid.You may run BLASTn homology search of the primers against E coli genome to check if it binds to the bacterial genomic DNA. Rest you are expected to get very good amplification.
best of luck,
SD
Update : I got the smaller fragment (1.2kb) amplified, and it checked out at the right size when run through the gel. I am still having problems getting the 4kb fragment to work. I am using phusion taq and GC buffer. I got two bands this time but they are around 2kb. So I must have had the primers grab something from the genome and not the plasmid? I will probably try now an alkaline lysis extraction and work from the pure plasmid. The primers I used came from the NEB primer designer. Here is an image of the gel I just ran, with the 2kb band. I wish I could sequence it!
I suggest that you perform plasmid extraction from the E.coli culture to obtain pure plasmid which you can then use as template in your PCR. Ensure that the primers you are using are specific to the plasmid you want to amplify.
I am going to do an alkaline lysis plasmid extraction today, as you recommend. I suspect something in a looped plasmid is tangling with something in the genomic material. Odd that I got such clean, matching bands though.
I was mystified by nice sharp bands, all at the wrong size. Then I double checked the plasmid sequence and found one of my forward primer sequences appears TWICE (one time in each fragment I was amplifying) due to multiple ori present. So now I am going to digest the plasmid and run separate PCR reactions on the fragments after gel separation.
Hi Amy,
Our following article can help you a little bit with the plasmid issue.
https://link.springer.com/article/10.1007/s40291-016-0195-2
Best wishes
SD
This is a classic rabbit-hole problem of forgetting to make absolutely sure all the knowns are known. This is what i would do before;
1. Find the complete plasmid map.
2. Even if you do find that, if it is possible just do an e.coli plasmid prep and send that in for sequencing using an established primer set, if this is a common commercial vector. Ideally you have 1 or two primers sets in your actual CDS/ORF.
3. Use your sequence data to fill any gaps on what maybe isn't congruent with what you are getting in your PCR and the expected plasmid map.
4. If needed, design primer sets that would result in a specific product size, specifically a product that covers your region of interest and a plasmid specific region, like the antibiotic resistance cassette.
I am reading literature that shows that linearized plasmid DNA is 9x more sensitive to PCR than circular DNA due to supercoiling plasmid topology. So I am linearizing ;-)
Not just plasmid DNA, linearized of bacterial and circular viral DNA also improve PCR performance greatly.
Hi,
Prof JOhn and Prof. S Dutta are accurate. You may directly perform a PCR and obtain amplification.
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