5 replies to this topic

[Wonyong]

Hi.


I already posted once about this before. I tried every answers replied onto it but still suffer the same issue.

The picture below is my headache.



It is an overnight E coli culture in LB+Ampicillin 100ug/ml. After transformation, I picked single colony on Amp plate and cultured. Next day the curture turns like this. No DNA recovered.

I've changed LB media, plate, even washed DNA with PCIAA. None seems contaminated.

If someone saw something like this before please let me know how to get rid of this issue.


Thanks.

[perneseblue]

It appears to me that there is little or no growth in the culture

There are several possible causes,
1- the colony that was selected does not have resistance to Amp.
2- there is some leftover detergent on the surface of the glass vessel.
3- contamination of the glassware by lytic phages.

1 - pick another colony.

2 - After the initial wash, soak/rinse out your glass ware in warm water. Then rinse 3 times with distilled water

3 - expose the glassware to UV for 10 minutes and that should denature any viral DNA.

[microgirl]

It does look like your culture didn't grow. Amp can be funny. Try going up to amp200 plates and see what happens.

[Wonyong]

There are a little growth of bacteria in that culture.

In fact, what I concern more than low growth level is that there are strange aggregates in the culture.

They appear in any cultures I grow regardless of glasswares, media, or DNA I transformed.

Is it possible that lytic phage induces such clots in the culture?

[aimikins]

wouldn't it most likely be some sort of ubiquitous fungus?  assuming you've already switched batches of LB?

[ah6tyfour]

I've seen this twice:

First time was when I was desperate to finish an expt but the transformation plate was empty except for a few hints of colonies forming (probably the start of satellite colonies).  So I put the plates back for 12 hours then picked those colonies.  They grew up exactly like that.  I still miniprepped them and had no DNA.

Second time was when I was in a rush to do a liquid culture.  So instead of taking the extra day to streak out a new plate from frozen stock, I scraped some cells from my month-old screening plate (you know...the plate you streak a little bit of your clone on before you inoculate your culture?).  They grew exactly like that and had negligible amounts of DNA.

The trend is unhealthy cells.  Make sure your amp plates are not too old.  If they're more than a month or so old, spread new Amp onto the plate before you go to dry the plates.

My bet is that what you are picking is not truly Amp resistant.  Are your colonies nice and plump and happy looking? (you know that feeling when you have a hard cloning reaction and you see a total of 6 colonies but they're so perky and plump you just know the have the clone? heh).

Transform 2ng of an empty plasmid, pick a colony, and see if that still happens.  And check to make sure your Amp stock is at the concentration you think it is.  Adding too much Amp will kill even Amp-resistant bacteria.

I also have clumping problems when I use XL-10 Gold from Stratagene.  For some reason that strain likes to clump on the bottom of the culture tube even shaking at 250-300rpm.  Every time I use that strain I see them clumping at the bottom of liquid cultures...which actually greatly decreases their ability to grow exponentially.  If I have two strains of bacteria and put both liquid cultures in the fridge for a couple hours, all the cultures with XL-10 Gold look like clear LB because all the cells stuck together and sank.  The cultures with the other strain will still be pretty homogeneous suspension.

Edited by ah6tyfour, 02 May 2009 - 07:55 AM.