Problems with growing out transformants in liquid LB - (Jan/09/2013 )

Hey,

So I've been working on a project for a couple of weeks and have been having an unusual problem. Right now we are trying to clone a DNA with a BAMHI/ECORI site into a PHC vector featuring the same restriction sites.

I have been able to grow out colonies on LB-Agar plates supplemented with Amp, however when picking colonies for inoculation, nothing grows out, or what grows out does not contain the DNA we want (ie primer dimers ligated into the vector.)

The plates do show signs of satellite colonies, however I figured if I chose the larger of the colonies (which I hope would have the desired insert) then the satellites wouldn't grow out and I wouldn't have to worry about it. However this is not the case. Most of the time nothing grows out, or has the sequence that is needed.

Any one have any suggestions? I do not believe the AMP concentration is a significant problem since our stock is at 5mg/mL. Although there are some precipitates that indicates that it was not completely dissolved. Even so, Isn't this concentration way more than I need anyway?

Also another thing to note about the plates is that they usually feature a lawn of colonies. From what i've seen, satellite colonies usually crowd around a larger colony, so as to grow within the region that has AMP degraded. However, from our plates it isn't clear (at least to me) which one is the satellite and which is the colony. I just assume the larger colonies are the ones I want, however, the others are maybe the same size.

Any suggestions?

Check whether Ampicillin is working properly or not.

If you are getting a lawn of colonies then you cannot clearly pick single colonies.

1) Are you plating a transformation or is this being streaked out from a glycerol stock?
2) If you are getting a lawn after transformation then you should plate less or plate a 1/10 and then the remainder (undilutied) on a second plate. This will help reduce the lawn your seeing.
3) if you are getting a lawn after streaking from a glycerol stock, then one of two things. YOur plates are contaminated (try incubating an empty plate O/N, see what happens) or your glycerol stock is contaminated.

Let me know what happens.

D

Well the lawn was only seen on 1 of the plates. There were sparodic clusters of colonies on various portions of the plate.

The Amp conc. is at 100microg/microL. I remade new plates and will get the results tomorrow so i'll update this with those results.

Also, The controls seemed okay: 1. No colonies on cells only or vector only ligation. And colonies were seen on an uncut plasmid at 1:1k dilution. However, skepticism tells me that some of the colonies on that plate were also satellites.