5 replies to this topic

[GCGGCCGC]

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

[Amy831]

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).

GCGGCCGC, on May 8 2009, 07:54 PM, said:

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.

[GCGGCCGC]

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?

Amy831, on May 8 2009, 11:02 PM, said:

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).

GCGGCCGC, on May 8 2009, 07:54 PM, said:

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

[Amy831]

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

GCGGCCGC, on May 8 2009, 08:26 PM, said:

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?

Amy831, on May 8 2009, 11:02 PM, said:

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).

GCGGCCGC, on May 8 2009, 07:54 PM, said:

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

[GCGGCCGC]

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.

Amy831, on May 8 2009, 11:48 PM, said:

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

GCGGCCGC, on May 8 2009, 08:26 PM, said:

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?

Amy831, on May 8 2009, 11:02 PM, said:

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).

GCGGCCGC, on May 8 2009, 07:54 PM, said:

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

[kajmak]

Just put them at 4 C.