11 replies to this topic

[epigenetics]

Hello,

i did a bad mistake 2 months back and i found that today.
Till now i stored the invitrogen top10 cells at -20 instead of -80. Yesterday i was trying to grow from stock and after 16 hours at 20 degree, the od is 0.035.
do you think the cells are bad and i have to throw all the cells away. or i can give more time and see whats happening.

Any suggestion will be appreaciated.

[bob1]

Your cells will not be well, and certainly won't be competent.  You either need to buy or make some more from a fresh stock of cells.

[hanming86]

Well stored E.coli probably will survive at -20C for quite awhile. i mean in 10% glycerol or so.

Try growing at 37 C with strong agitation

Edited by hanming86, 23 June 2009 - 05:09 PM.

[Curtis]

just a note from my own personal experience;

our -80 broke down 2 weeks ago and I had to put my chemically TOP10 competent cells in -20 freezer for a week before transferring to a new -80. I checked my cells yesterday after transformation and they work fine and I have many colonies on agar plate.

I save my cells in CaCl2-40% glycerol and I'm sure before transferring to -80 they were in unstable temperature, but they are still ok.

[hanming86]

Curtis, on Jun 26 2009, 12:35 PM, said:

just a note from my own personal experience;

our -80 broke down 2 weeks ago and I had to put my chemically TOP10 competent cells in -20 freezer for a week before transferring to a new -80. I checked my cells yesterday after transformation and they work fine and I have many colonies on agar plate.

I save my cells in CaCl2-40% glycerol and I'm sure before transferring to -80 they were in unstable temperature, but they are still ok.

yup. i dont have a -80 in lab n the downstair lab don work at odd hours. they been in -20 for awhile but still work.

[ram]

Once our -80 stopped working. Temperature went upto -10. Competent cells also failed!
You can still check efficiency of transformation with standard plasmid like pUC18 before discarding the cells.

[epigenetics]

ram, on Jun 28 2009, 06:06 AM, said:

Once our -80 stopped working. Temperature went upto -10. Competent cells also failed!
You can still check efficiency of transformation with standard plasmid like pUC18 before discarding the cells.

I talked to invitrogen, two aplication scientists said two different things, one said, it is trash now, other said, -20 might be ok,
so tried transformation with pUC plasmid,  got colonies, though they are not big, kind of small, but not too small, in my eyes it looks ok.

thanks

[ram]

Calculate the transformation efficiency now. Check out these site:
http://www.sciencega...s/transform.htm
http://www.sigmaaldr...efficiency.html

[epigenetics]

ram, on Jul 4 2009, 12:41 AM, said:

Calculate the transformation efficiency now. Check out these site:
http://www.sciencega...s/transform.htm
http://www.sigmaaldr...efficiency.html

thanks for these links,
I did with the 2nd one as the 1 st one is not working. The result is:

Transformation Efficiency:
1e4   Transformants / μg DNA or
1e1   Transformants / ng of DNA

Does it look ok?

[ram]

The efficiency appears quite low. For newly prepared cell in the lab, we get the it in the range between 1e6 and 1e7 per ng of DNA. So I feel either now you should not use these cells or use them only when you have large quantity of DNA available for transformation. Meaning, you should not use these for transforming ligation reaction where actual quantity of ligated product is quite low for getting sufficient colonies with the cells having such a low efficiency. On the other hand you can try using these cells if you have quite large quantity of plasmid available in your hand.
Regards
Ram

Edited by ram, 06 July 2009 - 09:48 PM.

[HomeBrew]

Why not just grow some of the cells, and make a fresh batch of competent cells?  Commercially-prepared competent cells are a waste of money, IMHO.  I can make ~30 ml of highly competent cells in a few hours following a protocol like this one.

[ram]

That is the best and safe option for experimenting with the bad cells