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freezing of E.coli culture prior DNA prep


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#1 GCGGCCGC

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Posted 08 May 2009 - 06:54 PM

Hi,

I was wondering whether anyone had tried to freeze E.coli prior DNA prep -and had success with the preparation? If so, what conditions did you use?

Thanks

#2 Amy831

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Posted 08 May 2009 - 07:02 PM

I'm not sure that I understand your question, but are you trying to freeze the E.coli following transformation?

If so, you could try a glycerol stock of the transformed bacteria, which works for us:
1 part bacteria (e.g., 500 microL) to 1 part autoclaved 80% glycerol (e.g., 500 microL), stored in a sterile cryotube. Make sure to tip to mix the bacteria into the glycerol. Immediately store at -80 deg C.

When you are ready to use, reinitiate a small volume (e.g., 50 uL) overnight in your growth broth (test out various volumes of the glycerol stock to see what volume grows up for you without overgrowing).

Hi,

I was wondering whether anyone had tried to freeze E.coli prior DNA prep -and had success with the preparation? If so, what conditions did you use?

Thanks


Edited by Amy831, 08 May 2009 - 07:12 PM.


#3 GCGGCCGC

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Posted 08 May 2009 - 07:26 PM

Once in a while, my E.coli cultures grow much slower than expected -like tonight. Instead of spending even more hours in the lab, I would prefer to store the bugs until coming week and to process them then. Any ideas?

I'm not sure that I understand your question, but are you trying to freeze the E.coli following transformation?

If so, you could try a glycerol stock of the transformed bacteria, which works for us:
1 part bacteria (e.g., 500 microL) to 1 part autoclaved 80% glycerol (e.g., 500 microL), stored in a sterile cryotube. Make sure to tip to mix the bacteria into the glycerol. Immediately store at -80 deg C.

When you are ready to use, reinitiate a small volume (e.g., 50 uL) overnight in your growth broth (test out various volumes of the glycerol stock to see what volume grows up for you without overgrowing).

Hi,

I was wondering whether anyone had tried to freeze E.coli prior DNA prep -and had success with the preparation? If so, what conditions did you use?

Thanks



#4 Amy831

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Posted 08 May 2009 - 07:48 PM

Yes, then use a glycerol stock (see instructions).

Once in a while, my E.coli cultures grow much slower than expected -like tonight. Instead of spending even more hours in the lab, I would prefer to store the bugs until coming week and to process them then. Any ideas?

I'm not sure that I understand your question, but are you trying to freeze the E.coli following transformation?

If so, you could try a glycerol stock of the transformed bacteria, which works for us:
1 part bacteria (e.g., 500 microL) to 1 part autoclaved 80% glycerol (e.g., 500 microL), stored in a sterile cryotube. Make sure to tip to mix the bacteria into the glycerol. Immediately store at -80 deg C.

When you are ready to use, reinitiate a small volume (e.g., 50 uL) overnight in your growth broth (test out various volumes of the glycerol stock to see what volume grows up for you without overgrowing).

Hi,

I was wondering whether anyone had tried to freeze E.coli prior DNA prep -and had success with the preparation? If so, what conditions did you use?

Thanks



#5 GCGGCCGC

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Posted 08 May 2009 - 08:04 PM

Thank you, Amy831! I will try that out. Before I would extract DNA from the stored E.coli, I would thaw them on ice and then wash out the glycerol with fresh media. That should work.

Yes, then use a glycerol stock (see instructions).

Once in a while, my E.coli cultures grow much slower than expected -like tonight. Instead of spending even more hours in the lab, I would prefer to store the bugs until coming week and to process them then. Any ideas?

I'm not sure that I understand your question, but are you trying to freeze the E.coli following transformation?

If so, you could try a glycerol stock of the transformed bacteria, which works for us:
1 part bacteria (e.g., 500 microL) to 1 part autoclaved 80% glycerol (e.g., 500 microL), stored in a sterile cryotube. Make sure to tip to mix the bacteria into the glycerol. Immediately store at -80 deg C.

When you are ready to use, reinitiate a small volume (e.g., 50 uL) overnight in your growth broth (test out various volumes of the glycerol stock to see what volume grows up for you without overgrowing).

Hi,

I was wondering whether anyone had tried to freeze E.coli prior DNA prep -and had success with the preparation? If so, what conditions did you use?

Thanks



#6 kajmak

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Posted 09 May 2009 - 01:27 AM

Just put them at 4 C.




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