I amplified a plasmid and now I have an eluted stock from a maxi prep i did. I sent the plasmid out for sequencing and it came back as no priming. I tried a restriction digestion with 2 different enzymes that should cut the vector in 2 places but all i have on the gel is one band of the uncut DNA.
The enzymes are still good because they cut other vectors. The DNA quality measurements are 260/280 = 1.9, 260/230 = 2.39.
This is very strange, I just want to confirm I have my plasmid. What could have gone wrong?
Hi Justine,
Sounds like the plasmid you isolated is not the one you want since the primer(s) you tried using gave you no sequence and the plasmid did not cut with 2 enzymes that should have cut. Did you use specific primers or common primer sequences that are found on most plasmids (M13, T7/T3, etc...)?
Where did you get the plasmid/bacterial clone you wanted to amplify? Did you transform the plasmid and then pick an isolated colony to amplify? If this is the case you may have had a contaminating plasmid get into your transformation from somewhere and you picked that clone. You can just pick a few more clones and do minipreps (keep a little of the culture aside) and verify your plasmid before doing a maxiprep.
If the plasimd came from a bacterial stock then that stock may be wrong/mislabeled.
Hi, I got the plasmid from Promega and used the suggested sequencing primer in the data sheet. I just wanted to make a larger stock of the plasmid for myself, so i took a small amount from the original vial, transformed bacteria, picked an isolated colony, and grew it up in the presence of the selection marker, carbenicillin. I was careful not to let the cultures or plates incubate too long to prevent satellite colonies. I don't know how a contaminating plasmid made its way into my prep but if it did it must have been ampicillin resistant...
Hi Justine,
I know that most commercial plasmid are sold in a linear form. They get circular only after ligation. According to what you wrote, it seems like you did perform a ligation step before transformation. This might be the reason the PCR with the sequencing primers is not amplifying anything.
thanks for the insights..I have decided I will just redo it...pick a new colony, grow it up, isolate it, and then sequence it. That way I will be sure I have my plasmid before I transfecting and possibly wondering why I don't have a signal.
I think the plasmid that you extract is different with plasmid you want and may you transformed another plasmid .
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