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pUC4K HincII sites, how many?

Molecular cloning plasmids microgiology genetics

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#1 Baroudeur

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Posted 28 August 2011 - 10:46 AM

Hello,

I am trying to cut out the kanamycin cassette from plasmid pUC4K with HincII. I have downloaded a corresponding sequence from Genbank and using VectorNTI, I can only see two HincII sites upstream and dowstream from the cassette, wich makes the enzyme perfect for my purpuse. Nevertheless, after migrating on gel I repeatedly get 3 bands rather than two. So does pUC4K has 2 or 3 HincII sites?

Thanks

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#2 pDNA

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Posted 28 August 2011 - 11:52 AM

pUC4k should only have two HincII sites! ...what is the size of the bands you see? have you ever loaded uncut vector next to your digest vector? ...maybe one of your bands corresponds to the supercoiled form and your vector is only digest partially? ...if so you'll have to pronlong incubation or increase enzyme concentration.

Regards,
p

#3 Baroudeur

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Posted 28 August 2011 - 12:21 PM

I'll do that tomorrow to rule out partial digestion ... and then I'll update the thread.

thx
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#4 Baroudeur

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Posted 29 August 2011 - 12:40 PM

Plasmid digested, but didn't have to migrate it today ... I'll migrate it early in the morning tomorrow (european time) we'll see ...

Baroudeur
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#5 Baroudeur

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Posted 30 August 2011 - 03:39 AM

and the long awaited image ... pUC4K has an undescribed HincII site right?? the only other explanation might be that I'm using a mislabled plasmid ...

Attached Thumbnails

  • pUC4K.JPG

Edited by Baroudeur, 30 August 2011 - 04:23 AM.

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#6 phage434

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Posted 30 August 2011 - 03:51 AM

Yes, it looks like it. The only other possible explanation would be uncut plasmid, which is why pDNA suggested running undigested plasmid along with your digestion. Is the sum of the fragment lengths the expected length of your plasmid? Can you cut your plasmid with a single site cutter?

#7 pDNA

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Posted 30 August 2011 - 03:52 AM

what i meant was put uncut vector next to digested vector on the same agarose gel ...then you would see if one of the bands corresponds to one of the uncut bands.

Regards,
p

#8 Baroudeur

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Posted 30 August 2011 - 04:32 AM

yeah guys I know, yesterday I had the chance to run the uncut vector alone but today I forgot to run it along the digest, the drawback of doing many things in parallel. Anyways I'll try to find yesterday's image, and I tend to think this isn't really uncut vector because I gave the enzyme enough time to cut and used 1µl of enzyme for every microgram of DNA ... anw I'll go migrate the uncut plasmid alone along with a ladder (SmartLadder 10kb in the image above).
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#9 pDNA

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Posted 30 August 2011 - 12:18 PM

when i look closely at your pic i see a ~1000 bp, a ~1500 bp and a ~2000 bp band ...to sum up 4500 bp ...pUC4K should be around 3900 bp ...the original bands should be around 1200 bp and 2700 bp ...this corresponds to the two upper bands, so it seems you have some 1000 bp additionally... possibly something is wrong with your vector ...have you checked the identity by digesting with another restriction enzyme? ...maybe it is already pUC4K with some insert?

Regards,
p

#10 Baroudeur

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Posted 31 August 2011 - 10:46 AM

P, this is what I suspect: that the petri dish where I'm subculturing the vector from might be mislabled. Have you digested pUC4K with HincII before?
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#11 Baroudeur

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Posted 31 August 2011 - 10:48 AM

btw, to be exact and complicate things more, the bands are @:

1000bp
1250bp
1750bp
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#12 pDNA

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Posted 31 August 2011 - 11:14 AM

to be honest ...never worked with pUC4K

another explanation would be a third HincII site introduced by a point mutation since HincII has a degenerated recognition site ...would be the only logic answer when you are sure your bands add up to the right size of 3.9kB (i'm not really used to your marker ...so you will know best what size the bands are).

Maybe the vector was subcultured to often ...mutations do happen ...i remember a case in our lab where we bought a plasmid from a vendor that starts with Clone and ends with tech ...the nuclotide sequence of the vector said that a certain restriction site was present and we were not able to cut the vector with that enzyme ...in the end we we proofed by sequencing that the original plasmid we bought had a point mutation that leads to the loss of the restriction site. It's biology :)

Why do you want to remove the Kan resistance?

Regards,
p

#13 Baroudeur

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Posted 31 August 2011 - 11:46 AM

I need it to replace a gene ...
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#14 pDNA

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Posted 31 August 2011 - 11:59 AM

no alternative restriction sites?

#15 Baroudeur

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Posted 31 August 2011 - 01:05 PM

Check this old article out, it has a map of pUC4K with 3 HincII sites, amazing! but not detailed enough, alas.

BROOME-SMITH, J. K. (1985). "Construction of a mutant of Escherichia coli that has deletions of both the penicillin-binding protein 5 and 6 genes." Journal of general microbiology 131(8): 2115.
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