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Transforming bacteria with plasmid from filter paper, months later

plasmid transformation filter paper room temperature

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5 replies to this topic

#1 Felicity185

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Posted 18 September 2011 - 09:00 PM

I was kindly sent a plasmid from an overseas researcher, successfully eluted using ddH2O and transformed bacteria, getting many colonies. However I was promptly told by my supervisor to drop everything I was doing and focus on writing my thesis. I put the tubes on my supervisor's lab bench for him to 'look after'.
It's now 7 months later and the DNA/paper/water tubes have been sitting at room temperature the whole time. I tried transforming bacteria again and only got colonies for one of the constructs (I have 3 constructs total).

So my questions are, what has happened to the DNA? Has it degraded (since it was just sitting in water at room temp), and is there a way i can rescue it (possibly by precipitate it before transformation?). I transform by heat shocking the bacteria... should I add an incubation in non-selective broth after the bacteria have cooled down again, to give them a chance to grow, before spreading on selective plates? Please help!
Felicity

#2 leelee

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Posted 18 September 2011 - 09:50 PM

Did you elute the DNA from the entire piece of paper? Or only some of it?
If the later, I would elute some more and try a transformation with that.

And yes you should DEFINITELY allow your bacteria to recover in non-selective broth before plating. You need to give them time to express the resistance genes that allow them to survive your selection agent.

#3 leelee

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Posted 18 September 2011 - 09:55 PM

And yes, there is a good chance the DNA has degraded- and you can't rescue it after that. If you are storing DNA long term, much better to do so in TE buffer to help prevent hydrolysis of the DNA.

Having said that, as a last resort, I wonder if it would be worth precipitating what you have and resuspending in a smaller volume. There may be enough non-degraded copies of the plasmid left to get a successful transformation when you concentrate the DNA?

Perhaps somebody more expert on plasmids and DNA degradation can tell me if this is realistic or if I am just being silly?Posted Image

#4 Felicity185

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Posted 18 September 2011 - 11:50 PM

Thanks for the reply leelee.
Unfortunately I eluted from the entire spot of DNA.

Only once or twice have I allowed the bacteria to recover in non-selective media prior to plating them, and most other cloning/subcloning that I've done has worked fine without that step. I will do that next time so that they have every opportunity to survive. I will also try precipitating it. But I'm hesitant to use all of my elution. I think I might add several hundred microlitres more water, mash it up some more and then aim to use half to three quarters of that volume for the precipitation step. Fingers crossed!

#5 phage434

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Posted 19 September 2011 - 05:04 AM

PCR is much more likely to recover DNA than transformation. As a last resort, you can design primers and clone the insert into new vector.
You can also try a "miniprep" of your transformed cells. There might still be plasmid in the cells. Make sure you are using high efficiency competent cells.

#6 allynspear

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Posted 23 September 2011 - 06:06 AM

I agree wholeheartedly with phage434 about PCR. You only need one lesion in a plasmid to make it non-viable for transformation, but you can get past that using multiple PCRs to recover the portion of the construct that you are most interested in. If this is not a viable alternative, I would purchase the highest efficiency competent cells you can find (XL-10 Gold would be a good shot) and do 5-10 transformations (do not pool competent cells, do 10 separate reactions) and plate out everything. This may mean you have to do 40 spread plates per construct but it's worth a shot. XL-10 gold cells can actually transform linear DNA with a decent efficiency which is not something that many other competent cells do well, so if you have lesions in your plasmid, you may be able to still transform it and have the XL-10s do the repair.

I would definitely not precipitate the DNA, since you may already have such a low concentration of viable plasmid that you will loose the rest of it trying to concentrate it.

As for outgrowth of your transformed cells, I understand that this is common practice, however this is only NECESSARY with bacteriocidal antibiotics, ie antibiotics that will kill the bacteria rather than just stop growth. Ampicillin is bacteriostatic, so it won't kill the bacteria, just stop them from growing. You can do a recovery or outgrowth step with ampicillin selection, but it is not essential since the bacteria will just sit on the plate not growing until they have made enough of Amp resistance gene product (beta-lactamase). For Kanamicin, however, if you do not give a recovery outgrowth, the bacteria will not have enough time to make the Kan resistance gene product (NPTI or II) and they will die in the presence of the antibiotic.

Best of Luck.





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