Ligation worked - maybe? - Aimikins - I've got colonies! Which size do I pick? (May/03/2006 )

Ok after much helpful info from everyone on this forum, esp. Aimikins (thanks again!) I have colonies from my ligation reactions! wohoo!

I plated 100ul of a positive control and am seeing 100+ colonies.
I plated 500ul of my negative control and got 4 colonies (uh-oh?)
I plated 500 ul of my ligation reaction and am seeing 20-50 colonies on average in my 4 plates.

Even though I got 4 colonies on my negative plate can I assume that some of the colonies on my ligation plate did work and they contain the insert?

I figure I'll just have to pick a load of colonies and do many many mini-preps and digest them all (and keep my fingers crossed?)

Some of the colonies are small and some are big - I've heard in the past that the smaller colonies tend to be the ones with the inserts? Is that correct?

I'm just so happy to see some colonies at this stage!!!

I personally wouldn't assume colony size is a good indicator of contents...some little colonies are just satellites...we all have our own theories in this regard, but I try to pick smorgasbord of colony size and try to get as many as I can

so if I am reading you right...500ul neg control (vector + water + ligation stuff, right?) gave 4 colonies

500ul ligation (vector + insert + ligation stuff) gave ~100 colonies spread over 4 plates..is this what you mean?

this means that approx 24/25 colonies on your ligation plates are not vector background..but this is always a rough estimate.

Again, everyone has a philosophy and strategy for such things, but if I were you I would pick about 50 for posterity (and also those 4 negatives)

I would miniprep 2 negatives and 10-20 (depending on your centrifuge size; I miniprep in batches of 18 because that's how many tubes my ufuge holds and it's convenient) of the ligation mix colonies and see what you get. retain the others in case you have to screen more

but that is just MHO. yay! good for you and I hope your colonies are the right thing

yep, 500ul of my negative plate (vector+ligation stuff+water) gave 4 colonies

500 ul of my ligation reactions (vector+insert+ligation stuff) gave:
plate 1 - 36 colonies
plate 2 - 18 colonies
plate 3 - 40 colonies
plate 4 - 40 colonies

didn't think to pick the negative colonies but will pick 2 to see what it is - I suspect they are WT plasmid where one of the REs didn't fully cut and it just re-annealed

I've picked 10 colonies from my ligation reactions but will go ahead and pick 10 more just in case - I figure I'll be mini-prepping all day tomorrow (have other ligations that worked) so what's another 10 added to that? ha ha

good luck, sounds like your numbers are good anyhow

I would colony PCR fourteen from the hundred colony plate, fourteen from the fifty colony plate, and include a no template control.

I would mini-prep four to eight of the colonies that showed up positive.

Mini-preps (about a $1.00 each) are far more expensive than colony pcr (about .17 per reaction).

-Matt