XL-10 Gold Cells at 42 Degrees
August 29, 2009 2:10 PM   Subscribe

What happens if I grow XL-10 Gold e coli at 42 degrees C?

Our warm room is acting screwy and maintenance can't come to fix it until Monday. I, however, was hoping to get some maxi-preps done this weekend so that I can submit for sequencing first thing Monday morning.

What will happen to my cells if I grow them at 42 degrees? Will it work? I have like 12 preps to grow so if it doesn't work out it's fairly wasteful, otherwise I'd just give it a shot to see.
posted by sickinthehead to Science & Nature (5 answers total) 1 user marked this as a favorite
 
When you transform your XL-10 gold competent cells, you subject them to a 'heat pulse' of 42 degrees to facilitate the entry of DNA into your cells.

Probably not a great idea to grow this particular strain at 42 degrees for optimal plasmid production. You could waste your time and materials, and any results you got from plasmids grown at the higher temperatures would be dodgy.

If you are in an academic setting, do you have any friends in adjoining labs? Maybe they have some space in their warm room. If you don't know anyone, use your problem as an icebreaker. You will get the cultures grown for your plasmid preps, and access to additional resources for the next time you're in a jam.
posted by Seppaku at 2:49 PM on August 29, 2009


I know that you heat shock at 42 degrees, but you also do so in the presence of BME, which I assume goes a long way in putting holes into the cell membrane.

I agree that it might not be optimal for production, but why would any DNA results be dodgy?
posted by sickinthehead at 2:54 PM on August 29, 2009


It's not so much the BME (though it helps) as much as the CaCl2 and the heat shock.

Dodgy because at 42 degrees, you're growing your XL-10s in heat shock mode. At that temperature, your bugs will be more concerned with making proteins to save their asses than with making your plasmids. Mistakes happen, especially when the e. coli aren't concentrating on the task at hand.
posted by Seppaku at 3:00 PM on August 29, 2009


What are you using as a culture vessel? Could you set up some kind of water bath or some such or are you using something to advanced for that.

You know what else is 37°...I've incubated a very small BCA assay in my pants pocket before to see if a real assay was worth doing or if, as I suspected, my protein A column was dead.
posted by Kid Charlemagne at 5:43 PM on August 29, 2009


Kid Charlemagne: good idea, but unfortunately I'm growing 400mL cultures in 1L flasks.

I decided to use the wonky warm room as an excuse to take the rest of the weekend off. :c)
posted by sickinthehead at 7:32 AM on August 30, 2009


« Older Help me be a good speech-giver tomorrow. Ready --...   |   Basic Black Underwear Newer »
This thread is closed to new comments.