Hi Piotr,

it is ok to assume that your plasmid is made up from fragments that sum up to the total length.

From the picture, plasmid 1 and 3 have an almost identical XbaI fingerprint, but a very different one with EcoRI.

2,4 & 5 have 'dirty'' fingerprint. Thus it 's hard, to sum up, the length of every fragment.

Good luck,

Nicolas

Yes, your XbaI digestion for the #1 and #3 yielded ~23-kb and ~10-kb bands.

It is hard to say whether the uncut one is 33 kb, because there is no DNA markers at this size on the gel.

@ Piotr Majewski:

What is the purpose to digest this 'unknown' plasmid with XbaI? Will the band pattern (23 kb + 10 kb) tell you what plasmid this is?

@ Yuan-Yeu Yeu

I am currently aiming at characterization of antimicrobial resistance vector im clinical bateria. Therefore I will attempt to describe its (1.) size, (2.) incompatibility / plasmidMLST, and also (3.) to confirm the presence of particular gene on purified plasmid.

PS at this stage I will not apply plasmid sequencing

@ Piotr,

What method your are going to use to confirm the presence of particular resistance gene on the unknown plasmid?

@ Yuan,

there is some other reliable way than Southern Blot followed by using gene-specific probe?

Are you talking about finding out the particular resistant gene? Yes, SB is a good way to go, but first you have to know the existing resistant gene and its sequence, so you can prepare your gene-specific probe. Do you know this particular resistant gene (and its gene sequence)?

Resistance gene is inserted info class I integron which is already sequenced. in your opinion, there is no other way than SB + probe hybridization? 

You can use PCR. But SB is safer for confirmation. PCR can be a false result due to contamination.

Since it is a resistant plasmid, can it be transferred into some empty competent cells and be selected on selection medium?

28

Hi Piotr

There are many causes and You can read some references in attachment 1

https://msu.edu/course/lb/145/luckie/inquiriesS2005/streamII/hurst/

2

https://msu.edu/course/lb/145/luckie/inquiriesS2005/streamII/wayo/

3

https://essayonrequest.com/an-introduction-to-the-identification-of-an/

4

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3837812/

5

http://www.microbiologyresearch.org/docserver/fulltext/micro/113/1/mic-113-1-73.pdf?expires=1492284723&id=id&accname=guest&checksum=92E28E952917B02847199AF5D512F97B

6

https://www.terrificscience.org/lessonpdfs/ControlledExp.pdf

7

https://ijbiotech.com/article_7010_941a045f17ea66a7c6c212607da5d410.pdf

8

http://www.chem.purdue.edu/teacher/table_of_contents/Gel%20Electrophoresis/PLSMD_LB-96.pdf

9

https://books.google.com/books?isbn=0803629753

10

http://sites.psu.edu/eportfoliomculver/files/2013/04/Using-Genes-for-Antibiotic-Resistance-to-Trace-Sources-of-Bacterial-Contamination.docx