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Plasmid digestion

Anonymous Sat 22 Sep 23:58:11 2018 No.10022735 View Reply Original Report

Quoted By:

Can someone help me out? Are my results normal?I feel like my double digestion is missing bands. Also is the band labeled F1 even a band?

From what i know the plasmid is pUC19, DNA1 is the parental plasmid while DNA2 and 3 are recombinant. Also the parental plasmid has only one EcoRI site and only one XbaI site.

Anonymous

Anonymous Sun 23 Sep 2018 00:14:26 No.10022758 Report

Quoted By: >>10022918

I think I understood, but please tell me if I'm wrong...

DNA2 & DNA3 are the same plasmid but with the insert in opposite orientations

The recombinant plasmids were made by digesting both the plasmid and the insert with EcoRI, so two EcoRI sites are created at both ends, which is why it has 2 EcoRI bands.

The insert probably contains an XbaI site as well which is why we get 2 XbaI bands. It's probably used to determine the orientation of the insert. Also, the distance between the EcoRI and Xba1 site is less than 30bp so there is probably another band in the double digests all the way at the bottom that can't be see & the F1 band is just a stain

Anonymous

Anonymous Sun 23 Sep 2018 01:58:52 No.10022918 Report

Quoted By: >>10022987

>>10022758
i agree, backwards insertion seems likely. OP, you did cut and paste, right? this is why you always test more than one colony.

OP, how big is your insert? also, why are you calling B2/B3 multiple bands? you'd only get that if your insert was as big as puc19 in the first place. is it?

personally i think F1 is a real band, something wild might have gone on with that plasmid. OP might have gotten multiple insertions.

Anonymous

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Anonymous Sun 23 Sep 2018 02:57:52 No.10022987 Report

Quoted By: >>10023030

>>10022918
Yep.

I don't know the exact length of the insert but according to my data it is as long as pUC19.

Anonymous

Anonymous Sun 23 Sep 2018 03:25:48 No.10023030 Report

Quoted By:

>>10022987
oh huh, weird

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