Stock not growing. Can i re-grow Plasmid from sample? - (Jul/04/2010 )

I've a follow-up question.

I found at the back of the fridge that i had kept the cells i made spread plates with before. If i have these ecoli bacterial cells on an agar plate, how long will they last in a fridge?
There's kanamycin antibiotic added to the plate, but the plate wasnt covered in tinfoil, so that might be degraded by now.
The plate still looks ok though. im just wondering if the cells would be viable or useful by now.

cm13 on Jul 12 2010, 12:29 PM said:

So to follow up on this:

1) I tried transforming my bacteria with my plasmid sample. My protocol was as i mentioned earlier. With 50ng of plasmid and 50ul of cells. I also changed the amount of SOC added to 1ml instead of 500ul. Having plated this, i left the plates at 37C overnight. There is nothing on the plates. Should i maybe leave it for another day?

2)I also found i had plates from the last time i made the plasmid. However these plates are 4 months old and were kept in the fridge. I attempted to take a colony from the plate, add it to 5mls of LB broth, and add 5ul of Kanamycin. (Again at a concentration of 30mg/ml).
Leaving this starter culture for 8 hours rotating at 37C , i found nothing. I left it overnight at the same conditions and there was still nothing.

Should i maybe try step 2 again, but without the kanamycin? This might allow for growth of the cells.

like the others said, try both of these:

heat shock transformation (my protocol)

thaw chemically competent cells on ice
add 1uL of plasmid (around 10 ng is fine) and mix gently with pipette
leave cells on ice for 30 min
heat shock cells @ 42 degrees for 2 min
leave cells on ice for 5 min
add 900uL SOC / LB, recover @ 37oC with shaking for 1 hour.
spin down in microfuge for 2 min at max speed, resuspend pellet in 100uL
plate 100uL resuspension on agar with appropriate antibiotic, incubate 37 degrees overnight.

make sure to include a control transformation of another plasmid, so you know your compentent cells are working.

last shot recovery of glycerol stock:

innoculate entire glycerol stock in 3mL LB/SOC @ 37 degrees for a couple hours
as before spin down to collect pellet and resuspend in 100uL
plate on agar with appropriate antibiotic, also re-use the tube with LB/SOC + antibiotic and incubate @ 37 overnight.

remember to make multiple glycerol freezer stocks next time, I use 400uL of 50% glycerol and 600uL of culture.

the plate in the fridge could still be viable as well if its not dried out completely, so try innoculating big loopfuls into both media and media + antibiotic.

good luck!

Thank you everyone for the help.
I managed to get my plasmids growing properly in the end after transforming in my old plasmid into the ecoli cells.
I also made up new Agar plates with the colonies, and new glycerol stocks, so i should be ok for regrowing in the future.

Thanks once again!

Now would be the time to test your gycerol stocks.