Screening Tranformation Colonies: Funny Gel Pic and no plasmid?!?! - (May/19/2008 )

I got some funny DNA Gel Electrophoresis results and really need some guidance.

Gel of Bacterial lysate (DH5-Alpha cells lyed using SDS. Ran total cell Lysate)

Background:

In short, I inserted a 700 bp insert into a pET28b plasmid (about 5500 bp w/ Kanamycin resistance). I did a ligation and ran a gel of it. I then cut out the band that corresponds to the size of the plasmid+insert and purified it. Heat shocked to transform. Colonies grow very slowly and die if left on a plate for a week. Overnights don’t even get very cloudy (only a little)!

I lysed the cells with SDS, spun down debris, and ran the resulting liquid in a 1% TAE gel and stained with EtBr. Its basically total cell lysate...THIS IS THE GEL SHOWN ABOVE.

Funny Part:

No plasmid at 6,000 bp
Mystery ghost bands at 1,500 bp
Unusual (but NICE) bands around 12,000 bp
Purification of bands at 12,000 bp yield no DNA using MOBIO kit (3x)!
12,000 bp bands disappear when exposed to UV for 15 min.

My conclusions:

Could the 12,000 bp band be a mystery plasmid and I have contaminant bacteria?
Could the 12,000 bp band be RNA?
Could the 1,500 bp band be RNA?
Is the 12,000 bp band my desired plasmid in a weird configuration like supercoiling?


I'm out of ideas and would LOVE if someone could help me.

My goal is to get this plasmid w/ insert purified that is if my bacteria even have the plasmid I want!!!!

Please help.

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I'm quite confused why did you run your ligation onto a gel ? You should directly transform your ligation into the E.coli competent cells, there's no need to do such a procedure for cloning a 700bp into your plasmid

As for the gel it looks quite weird the 12kb band seems to be very condense and dosen't really looks like genomic DNA it cab be anither plamid but I doubt that you would get a contaminant if you use antibiotic selection to grow the colonies

As for the 1500bp it's surely not RNA beacuse tital RNA from bacteria generally runs at the bottom of the migration and not as a distinctive band

The bad thing is taht your culture doesn't grow nicely overnight taht sounds like a weird stuff is going on

Ok let's try to propose you a way out of theses tourments

Do your ligation normally, transform the bugs and plate them onto a LB agar plate with fresh antibiotics (Kanamycin 50 micrograms/ml)

Next day streak out if the colonies are too close to each other look carefully if all colonies have the same size

Start an overnight culture with prewarmed LB + kanamycin and inoculate with an isolated colonny
Perform a miniprep and purify the plasmid onto a column (Quiagen or Macherey Nagel spin kits)

Digest with the restriction enzyme used to clone the fragment (or adjacent enzymes if the sites are lost upon ligation)

As an alternative you can also perform a PCR reaction with specific primers wich are flanking the cloning region (if you have such like T7 and Sp6 or M13 universal primers)

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Hi there!

Basically, I'd say, follow Jipes suggestion.

Just another thought:

You didn't do a restriction digest, did you? But I assume your DNA ladder consists of linear DNA? So you really can't compare non-restricted DNA to linear.... Your DNA is possibly supercoiled or open nicked, meaning running faster or slower, respectively, through the gel than you'd assume it to. You should take this into concern...

Good luck!

Mike

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That's absolutely right it might just be the right plamid but undigested of course doesn't show the right size

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Thanks so much for the help.

Yesterday I cut out the 12,000 bp bands and purified them (MoBIO kit). I then restriction digested with NdeI and XhoI and ran this on a gel. SAW NO DNA at all.

Miniprep directly from the overnight cultures was attempted (6 times) and NO DNA is seen on the gel, just empty gels with ladder is all i get. It is possible that the solutions for Miniprep are bad, but the MOBIO kit has been really reliable, except for this screening! (I just don't know where to turn to anymore).

I cant get new supplies either because its a high school.

So either...


1.) My bacteria have no plasmid
2.) The 12,000 bp band is RNA and it hydrolyzes when purified with the DNA kit
3.) My GE Miniprep solutions are bad
---OR---
4.) God is trying to torture me

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Follow what Jipes recommended and tell us what happens.

The 100bp smear is most likely the only RNA on the gel.

How are you getting the materials for your ligation? Why are you gel purifying your ligation? Don't let your plates grow longer than overnight, even if the colonies are small. What is your Kan concentration?

I assume that when you use gel purified product, that you don't use the entire volume. It's very easy to dilute your DNA when gel purifying and as a result, makes it very difficult to re-visualize on a gel.

Are you doing a negative control for your transformation?

Short-wave UV exposure kills DNA.

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I don't think the Kit is the problem it's rather the bacterial culture, I had this once with a stroung contaminant which was overgrowing the real bugs. Mainly it's an antibiotic failure



First explanation seems the most promissing, second is simply impossible, third pretty unexpected but you can check by culturing and exctracting DNA from a known clone, Fourth explanation is "Life is like a box of chocolates you never know what you gonna get "

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Hi jeff and everyone,
I wonder if you have solved your mystery? especially the part that culture grow slow after O/N incubation, because I am facing the same problem here. I am doing a site-directed mutagenesis by following quick-change protocol from Stratagene. After DpnI treatment, I transformed reaction into GC10 competent cells, grow O/N on carb/agar plate. Examine the plate next day and the colonies look fine. Pick them out and grow in LB with selection at 37C, O/N, the culture look very dilute, and I only got a small bacterial pellet. Use Qiagen miniprep kit and got no plasmid at all.

Hope some help!!

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Slow growth normally imply 1 . ur antibiotic conc is too high or 2. ur culture got no plasmid ( some residual growth are sometimes expected especially when u use carbenicillin as ur antibiotic or any bacteriostatic type antibiotic). DID you do a control of " no plasmid" to check for the strength of your antibiotic. u DIDNOT mention anythiong about control. Experiment w/o control = experiment w/o direction

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Good idea!! But I think if I need a control to know if the [carb] is too high, I should transform bugs with the same vactor that I used to do the cloning. Well, I guess Experiment w/o logic = Experiment w/o brain.

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