John Forsberg

As I've never used those markers before, I'm not sure I'm confident of the band I'm pointing out, but if your markers are what I think they are I'd say your protein is the band in the induced total and soluble lanes that is just above the third obvious marker from the bottom.

I've attached the band I'm thinking is correct, and what I think the markers are.  I can't be positive, as I usually use multicolored markers, and I usually get a feel for how the markers run on a given gel type over time, so I can tell at a glance which markers are which.

I'll also note that with many protein expressions that I do, I do not see my protein in the total extract at all, but only see it after enrichment after binding to affinity resin and eluting (for many His-tagged proteins in particular).  So you may want to try a trial run of your first purification step to see if you get a more prominent band at the right size.

 12.10.2012 SDS UBC1 (1).jpg   20.11K   56 downloads

How long were the colonies on the selection plate?  Sometimes if the antibiotic is degraded near the colony, the colony can get rid of your plasmid and fail to grow.  That usually takes a substantial amount of time though.

Or maybe the plates are old?  Ampicillin is probably the least stable of the commonly used antibiotics in plates.  So maybe your colonies don't actually have your plasmid?

Another possibility is that your plasmid is expressing a toxic protein that is expressing enough in regular media to kill the cells after a little bit of growth.  Some media types (or commercial ingredients) can cause your cells to autoinduce after they reach a certain cell density.  Using catabolite repression for lac operon expression plasmids (adding 0.5% glucose or so) can prevent unwanted protein expression during your overnights.  If that might be a concern, then you can also look into Studier's autoinduction media papers/docs, for recipes that can suppress toxic protein expression.

We usually use a NanoDrop to quantify our DNA after cleanups/gel extractions.  I would normally hope to get ~50-100 ng/µl for a vector gel extraction, and maybe 20-50 ng/µl for my inserts (if 5 µl of the PCR product looks clean with a single band on the gel, I'll just do a PCR cleanup).  How did you quantify the DNA from your samples earlier?  You said you had "pcr purification and it is 138 ng/ul and 158 ng/ul. my vector is 6000 bp and 580 ng/ ul"?

For a ligation, I would mix roughly 100 ng total DNA of vector+insert in 10 µl total volume for the reaction.  For a first try, most people seem to suggest a ratio of 3:1 molar excess of insert over vector.  If that doesn't work, but you're sure your enzymes are cutting, you can try changing the ratio lower or higher (from 1:1 to maybe 10:1 insert:vector).

For that calculation:   ng insert = 3*(ng vector)(bp insert)/(bp vector)  

In your cloning, with a 200 bp insert, 6000 bp vector, I would use: ng insert = 3*(90 ng vector)(200 bp)/(6000 bp) = 9 ng insert with 90 ng vector.  (I roughly estimate the amount of vector to use by taking the length of the vector (6000) and add 3x the length of the insert (600) (total 6600), then figure out what % of the total the vector is (6000/6600 is ~90%))  There may be an easier way to calculate it, but this is how I do it.

I have also been told that if you run your DNAs on a gel, if you can see the band at a given volume loaded, then you can use that amount in a ligation (whether it is the insert or the vector).  So if you loaded 2 µl of your insert and vector DNAs, and can faintly see bands for both of them, then that's a good amount of DNA of each to try in a ligation.  I've never done my ligations that way, but if you have no way to determine the concentration of DNA in your samples, then that might be worth trying.

They mean that at least part of the assigment ("investigate") is to dig for information on your topic.  What you requested (at least sounded like) you wanted someone to just give you answers.

If I were you, I'd start looking through Wikipedia on hair color looking for what makes hair particular colors, what chemicals are in different hair dyes (or how they work), etc.  I'm newer than the others in the forums here, so I may be breaking a rule by even telling you to do that much.

In that case, I'd probably restreak, make sure the colonies looked normal, then grow up from there.  Even if you froze them down in media that originally had gentamicin (which will have degraded as the culture grew), I wouldn't rely on it being so clean that you won't get contamination when subculturing without antibiotics.  That sounds like a solid case for picking a colony for an experiment.  I realize that when using sterile technique people don't normally get contamination of their stocks, but you really (really) don't want that happening in an actual experiment.  Sometimes waiting a day is worth it.  If you were using antibiotics in your subcultures, then I'd say to not bother with the streaking.