Dear Researchers,
I performed conjugation via filter mating technique to transfer the Plasmid from Lactococcus garvieae to Enterococcus Faecalis (OG1RF).
The transconjugant were confirmed by the using selective antibiotics and further confirmation was performed by typing with Lancefield D antiserum.
I tried to extract the Plasmid DNA from conjugant by using Illustra plasmid prerp mini spin kit.
3mL of transconjugant overnight grown culture with respective antibiotics was cell down and the pellet was treated with 5oul of lysis buffer(50mg/mL lysozyme and 200 unit/mL mutanolysin in TE buffer ) incubated at 37C for 30 minutes. After that 25ul of 10% SDS was added in the sample. This Processed sample was than further treated as per instruction provided in the Kit.
Final elution was performed in 25ul of Elution buffer provided in the kit.
Nano drop value were found DNA, 55.5ng/ul, 260/280,1.87, 260/230,1.603.
I Loaded 12ul of this Pasmid DNA + 4 ul 6xloading buffer on the gel (0.6%) and run 25 minutes at 100 volt. I didn’t see any band.
We assume that Plasmid size is between 5-20 Kb, So in the gel 1st well 2.5 kb marker,2nd well genomic DNA and 3rdwell was the Plasmid DNa.
Please guide, how we can see the plasmid DNA band on the gel?
Is this a natural conjugative plasmid that you are referring to, or an artificial construct made in the lab. Natural conjugative plasmids can be very large, 30-200KB in size, and also are very low copy number. So a typical plasmid prep is not very effective with these types of plasmids. It may be that the plasmid is present but just in such low quantities you don't see it. Or if it was large, then it will functionally behave like genomic DNA.
On the other hand, if this is an engineered plasmid it probably is higher copy number and you should know what the size is. Again your yields might be low, so it could be as simple as needing more DNA in order to visualize.
We transfer a natural plasmid into a gram postive plasmid free strain via filter mating, thus assuming it a natural transconjugant.
How much plasmid Dna is necessary to visualize it on gel?
You can easily visualize 20ng (more is better). But the problem is that with large plasmids you won't get much DNA from a plasmid prep because it purifies with the chromosomal DNA.
I am agree with Benedik. I also have experienced when I want to collect conjugation plasmid harboring resistance gene from recipient cell by midiprep to confirm my conjugation, but it was failed. Finally I performed hybrid sequence by using Miseq and nanopore to confirm the resistance plasmid.
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