plasmid sequencing problem from single colony (mixed sequencing) (View forum version)



Biogareth

Posted 19 November 2011 - 04:11 AM

I sent one plasimd for sequncing from single colony which grew on M9 minimal medium plate. But when I got the results, there are one position (one amino acid) still containing mutation library (see in attachment). It is not logical because normally one colony only contain one kind of pure plasmids. or minimal medium has exception?
Could you give me some suggestions? Thanks!

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phage434

Posted 19 November 2011 - 07:43 AM

There could be multiple copies of your insert in a single plasmid. Both will sequence, and give you mixed readings at one position. I've had this happen once with a plasmid that was two entire copies of the expected plasmid, one with mutations in the origin that allowed replication (double origin plasmids are normally selected against). This plasmid also had a single position mixed base, similar to what you have found. I cut the plasmid with an enzyme, religated and transformed, and found correct plasmid in the transformants.

Biogareth

Posted 20 November 2011 - 02:46 AM

There could be multiple copies of your insert in a single plasmid. Both will sequence, and give you mixed readings at one position. I've had this happen once with a plasmid that was two entire copies of the expected plasmid, one with mutations in the origin that allowed replication (double origin plasmids are normally selected against). This plasmid also had a single position mixed base, similar to what you have found. I cut the plasmid with an enzyme, religated and transformed, and found correct plasmid in the transformants.


Thank you Phage434.
I am not sure I understand it correctly. Dose it mean that probably one of my plasmid has mutation at origin, and allow two more plasmids (mixed bases in my target gene) to replicate in one cell? Thanks!

phage434

Posted 20 November 2011 - 07:02 AM

In my case (not necessarily what is happening in yours) I had a single, double length plasmid with one active origin and one mutated, inactive origin. These carried two different sequences (similar to the trace you have above) that were impossible to resolve with any amount of streaking or re-transformation. Cutting with a single enzyme and religating and transforming gave a single length circular plasmids with a well defined sequence.