Rehydrating plasmids off of filter paper

[bluefly]

Hi! Thanks for the help. I have 3 precious plasmid samples that were mailed to me on filter paper. What is the best protocol for putting into solution so can transform into bacteria and make stocks etc...?

Thanks!

[MtDNA]

Simply place into a 15mL conical with the buffer (TE, TRIS-HCl etc.) of your choice.

[bluefly]

Thank you!

[relaxin]

Simply place into a 15mL conical with the buffer (TE, TRIS-HCl etc.) of your choice.

15 mL seem to be a large volume. I would use the minimal volume just enough to cover the filter, say 150 ul. I had a bad experience; the plasmid has such a low concentration that I could not transform it to 10^9 cfu/ug competent cells. I can only amplify the insert by PCR. When I send plasmids to other people, I mark the spot with pencil, so that they can cut out the spot (not the entire filter) and elute the DNA.

[bluefly]

Thanks Relaxin. I thought that might be a bit of a large volume also. I'm grateful for your added advice!

[mpagel]

GENTLE heating may be called for as well. GENTLE = no boiling or microwaving!!!

42 degrees C max.

[MtDNA]

I didn't say place it in 15mL of buffer, I said place it into a 15mL conical. I guess I presumed the OP knew to use a volume of buffer consistent to their needs. To even think of using 15mL of buffer is ridiculous.

[bluefly]

Thanks for the help. Your wording was specific to putting it into a 15 mL conicle, and a volume was not mentioned, so no worries! I understood what you meant, just the volume to add is a bit arbitrary if you don't know the initial starting concentration, so an approximate idea of what volume people will generally use is helpful, so thanks again! Very grateful for all the input...

[relaxin]

I guess I am the only one who misunderstood the volume.

It all depends on the size of the filter paper. If you curl up a 3.5 cm diameter filter circle, stuff it into a 15 ml tube and put enough TE to cover it, the volume would have been 4 ml. It is still too much. You cannot believe that some people are so stingy and send only 1 ug plasmid on a 3.5 cm diameter filter circle.

I would cut up the filter and elute the DNA in a minimal volume of TE in a microfuge tube.

[mchlbrmn]

The competent bacteria I use have a transformation efficiency of >10^9 colonies/ug. So officially 0.1 pg in a transformation should give 100 colonies. So, in the real world ng's or pg's of intact DNA should give colonies, and it can be a good idea to plate a dilution if you predict it might be too crowded. Other than that it's just the practical matter of using a volume that won't be entirely absorbed by the paper. It's not rocket science, if you use too much DNA and it's a clone (not a library) it's not a big problem if more than one plasmid goes into a cell.

PS. I'm one of those stingy people. Remember with global warming, limited resources, recession, etc, why waste resources? I pipet into a circle marked on the paper so only a small volume is necessary. If I sent more than 1 ug it would be a waste to put it on paper when in a tube it could be used directly for a cloning.

[mpagel]

even if it's only 1uL, I prefer it to be in a tube. I can add my bacteria to the tube to do my transformation after having spun down the DNA to the bottom. If I don't see a droplet of DNA, I'll "wash" the sides of the tube with the competent cells (of course slowly and with only a single pipetting).

[relaxin]

I also prefer the plasmid in tube. But that will increase the cost of shipment. Of course, stingy people will simply put the tube inside a regular envelope. I once received a plasmid in a 200 ul PCR tube, and it got crushed during transit. Fortunately, I got some DNA out by rinsing the fragments of the tube.

[mchlbrmn]

Hmmmm, good idea. Perhaps I could save some money by crushing a tube, and using it to send plasmids for the next dozen requests.
If it's small enough, I could save more by e-mailing it.