Recovery plasmids from dead bacteria - How can i rescue my plasmid from dead cells??? (Jun/26/2009 )

Hi everyone... am new on forums and very much a new student, would appreciate any help plz

I have a little problem wondering if you can give me any advise.
My thrombostasin gene arrived from the USA in 1 eppendorph glycerol stock, ready transformed into a pTriEX-4 vector.
first thing i did, is checking the cell viability,
I checked it was an apicilin sensitive vector, so i made an agar an streaked the gel by scratching the surface of the iced cells in eppendorph on to the sensitive agar plate (amp 100ug/ml) so i put 100 ul into a 100 ml of agar.
Finally, incubating the plate at 37C overnight.

In the morning NO cells at all....

The packaging of cells was terribal (no dry ice) it cud be so... but is there anyway we can still rescue the Plasmid...anyway at all....??????

some alternatives that were discussed:- (in order)

1- leave them to grow more and see what happens?
2- PCR am not sure hows that gonna happen---they have some primers..bla bla bla
3- Do a miniprep on them to extract the plasmid but its likely to bring us nothing - since it is Rosetta cells?

Thanks

Ahmed


thanks

First off -- welcome to the BioForum, ahmed al jeffery!

What quantity of cells do you have in the Eppendorf from the USA?

You say you tried to recover the cells by "scratching the surface of the iced cells" and plating, but then you say the cells arrived with "no dry ice". Did the cells arrive frozen, or did you freeze them after you received them? If you froze them after recieving them, and there was no cryoprotectant in the storage media (e.g. glycerol), this may have killed the cells.

Have you checked viability on non-selective agar, or inoculated some non-selective liquid media, allowed this culture to grow for a while, and plated an aliquot of that culture that to selective agar? Maybe the cells need an expression period to overcome phenotypic lag. Not usually the case with ampicillin, but you should try it anyway.

If you can't recover the cells by either of these methods, I would do a miniprep on an aliquot of the cells you received and transform some competent cells.

I would also speak with the company or lab that sent you the cells and see if they have any insight into why you're having trouble.

In re-reading your post, you say:

ahmed al jeffery on Jun 26 2009, 05:59 AM said:

If the vector does not confer ampicillin resistance (you say it's an "apicilin sensitive vector"), these cells would not be expected to grow on ampicillin plates...?

An unmodified pTriEx-4 vector does contain the bla gene (see here), and so should confer resistance to ampicillin.

Thanks for your reply HomeBrew

I received 2x 1m of cells from the lab in the USA. It wasnt packaged properly, on receiving it, the samples were re-frozen in the -80C. for less than 2hrs. (The cell stock was already in glycerol)

Then, I used the stock to check cell viability, by streaking on amp+ sensitive plates and incubated overnight.

The o/n plates was almost crystal clear, with only a tiny growth millimeters wide on the very edge of the plates ( 2 plates).

Steps taken to recover our plasmid of interest:-

1- I incubates 5ul of stock into 2ml LB with NO AMP & incubated at 37C o/n

2- Using the small growth on 1st plate, i scooped the colonies to inoculate 2ml of LB and incubate at 37c o/n

3- To rule out problems with agar plate, i repeated to check for cell viability on different amp+ sensitive agar plates and incubated at 37c o/n.

I also left the original unsuccessful plates to continue growing in 37c

Thank you for your help HomeB

Ahmed

I would defrost one of the one milliliter aliquots, centrifuge it to a pellet, and do a plasmid miniprep on it. Then I would use some of the recovered plasmid DNA to transform competent cells, and some to visualize on a gel. If you have enough plasmid DNA, I would also do a restriction digest to try and bolster the ID of the plasmid (refer to the map of pTriEx-4).

For example, Bsa AI cuts pTriEx-4 three times, producing fragments of 1355, 2372, and 2965 basepairs. These should be resolvable on a gel. Remember, if your plasmid has an insert, you must account for it as well -- if you know the sequence of your insert, you can find a restriction enzyme that will produce a suitable digest pattern by doing an in silico digest.

Be sure to save sufficient material (probably the empty tube is enough) to do PCR with pTriEX-4 primers to recover the insert, if absolutely necessary.