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Removing unwanted sequence from pUC19


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4 replies to this topic

#1 seanspotatobusiness

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Posted 16 August 2011 - 02:56 AM

Here is a link to the pUC19 map provided by NEB. I don't need the lacZ. As far as I know, all I need is the ampcillin gene and promotor and the origin. If I cut with Ssp1 and AflIII, removed the fragment and inserted my own sequence of interest, would the plasmid function (replicate be selectable in bacteria via ampicillin)? I'd like the plasmid as small as possible.

#2 phage434

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Posted 16 August 2011 - 03:53 AM

You might want to look at the stripped down pUC19 plasmid we did for Biobricks, pSB1A3. It was made by PCR cutting out some of the unnecessary sequence, and works fine:
http://partsregistry...tle=Part:pSB1A3

#3 seanspotatobusiness

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Posted 16 August 2011 - 05:35 AM

Hi. According to that website, the quality control sequencing is inconsistent with the sequence it's supposed to contain? Do you know anything about that?

#4 phage434

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Posted 16 August 2011 - 09:40 AM

Ignore that. This is a result of sequencing using primers intended for the insert, rather than the backbone plasmid. This is sequence for the backbone plasmid, and it will never sequence "correctly" with those primers. We've been using this plasmid for 10 years with consistent results.



#5 prodes

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Posted 16 August 2011 - 11:32 AM

Hi,

If you are using SspI you will remove the promoter for the b-lactamase conferring the AmpR. Otherwise AflIII is in a neutral location not interfering with the origin of replication therefore giving green light.

Cheers




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