BAC DNA purification

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BAC DNA purification

Postby kiruphagaran » Mar 01 2018 10:32 pm

Hi all,

I am trying to purify BAC DNA (106 kb) using Qiagen large construct kit. I am able to elute 5 -10 μg DNA (measured by nanodrop) but when I run on agarose gel I see a a big chunk near the well (attached agarose gel pic). I contacted Qiagen technical help and they told it might be ecoli genomic DNA contamination. There is an exonuclease digestion step and I tried different exonuclease concentrations and incubation times but still I see the band near the well. Can anyone suggest me how to troubleshoot this problem and some other BAC DNA purification kit.

Thanks,
Kirupa
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Re: BAC DNA purification

Postby r.rosati » Mar 02 2018 12:27 pm

Hi!
What % agarose is the gel, and what ladder are you using?
Im assuming it's a broad-range ladder and the agarose is 0.8%... Yes, it does sorta look like gDNA, and I agree that the exonuclease could have removed it; but there seems to be so much of it, that perhaps the exonuclease wasn't enough. Sticking to the well isn't normal, though; if you use a ladder like Lambda DNA / HindIII cut, you'll see that even the 23kb band does leave the well, because at that size it just passes through the agarose as a thread oriented to the ends of the gel. I would expect even gDNA to have ran further than that, and left the well, unless it's precipitated, denatured (and it renatured in some 3D configurations that don't pass through the gel), or still bound to proteins (or the exonuclease). Weird.
I'm not saying it's your case, but gDNA contamination mostly happens when gDNA gets sheared during bacterial lysis, and it no longer precipitates out of solution together with cell debris. Are you by chance vortexing the lysed cells at the P2 and P3 stage, as opposed to gently mixing them?

Regarding other options, it depends on what you want to do. If the plasmid is for transfection and needs to stay circular/closed, I'd say that the kit you're using should be adequate... While if you don't mind the plasmid DNA being partially open, then you can broaden the range of options. Overall, in my opinion BAC purification differs from standard plasmid prep for three main reasons:
- low copy number means you need to purify from a bigger pellet;
- increased plasmid size might cause shearing in spin-column kits;
- the plasmid might also not elute very well from columns.

I did my PhD in a molecular cytogenetics lab, and we used to purify lots of BACs and PACs for FISH. We were "faithful" to the Qiagen Midi kit, which works by gravity flow too, and gave us good results most of the times. We used molecular biology grade glycogen as DNA carrier, and we made sure to heat the elution buffer at 55°C so that the plasmid DNA would come off the column more readily. We didn't have much bacterial DNA contamination, but we made sure to mix gently at the P2 and P3 stages.
I don't have direct experience with other brands for BAC purification, but... you could also try switching brands too.
Finally, try asking for a replacement kit from a different lot. Who knows, maybe the exonuclease from the kit is bad. Just because it's a kit doesn't mean that it's perfect :)
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Re: BAC DNA purification

Postby kiruphagaran » Mar 02 2018 3:09 pm

Hi Rosati,

Thank you very much for the reply and suggestions. Yes, it is 0.8 % agarose and the marker is 1kb O'GeneRuler (thermo scientific). I gently handle the p2 and p3 steps and avoid pipette tips through out the process. But when using the re suspension buffer p1 I handle it bit harsh. I also tried various exonuclease and ATP concentrations and incubation times but nothing seems to work. I also did a phenol/chloroform to remove protein contamination but still I see the band near the well. I heat the elution buffer at 65 C and use it. I am trying to do a dialysis and see if it can reduce that band and also try some other kits. As you suggested I will contact Qiagen for a replacement kit.

Thanks once again,
kirupa
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Re: BAC DNA purification

Postby kiruphagaran » Mar 02 2018 3:33 pm

I would like to add to the previous reply. The protocol says 1hr incubation with 200 μl exonuclease but when I increased the time to 90 mins and exonuclease to 220 μl I dont see the DNA but still the band near the well exist. I have attached the gel pic.
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Re: BAC DNA purification

Postby r.rosati » Mar 02 2018 5:02 pm

What if you're just seeing undissolved DNA? How many microliters did you load there?
I would perhaps tweak a few things for troubleshooting:
1) Leaving the DNA a bit more in buffer EX before adding the exonuclease: say, leaving it 1 hour at 55°C, then waiting it to cool at room temperature, and finally adding the exonuclease;
2) The final step already mentions leaving the DNA 1-2h @ 55°C in your buffer of choice, so I'd still do that, and *then* centrifuge 30min @ 16000g, and only collect the supernatant (if you see any pellet, save the tube with the pellet anyways, perhaps)? - then load the supernatant on the gel.

Also, if you want to test the exonuclease, you could take some EX buffer, add a known gDNA (doesn't need to be bacterial; e.g. salmon sperm DNA), and add exonuclease and proceed with the protocol; and check on a gel before/after exonuclease, to see if it works. But only if you can spend time doing these kinds of tests; otherwise I'd see if they give you a replacement, and then do an experiment side by side with the two lots.
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Re: BAC DNA purification

Postby kiruphagaran » Mar 02 2018 5:59 pm

Are you asking about the exonuclease I used in the preps shown in the first agarose gel? If so it is 200 μl. But I forgot to mention that I see a thin transparent material after adding buffer EX and it is not dissolving in EX ( I tried rotating for 2hrs at room temp) but when I measured the DNA concentration by nanodrop I have a lot of DNA (30 ug/mL). May be as you suggested I will incubate after adding EX buffer and see. I also think that it is undissolved DNA because when I incubate longer with exonuclease the band above 10 kb disappears but the chunk near the well remains.

Thanks once again,
kirupa
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Re: BAC DNA purification

Postby 29yrsExperience » Mar 12 2018 3:24 pm

If I may add to the fine suggestions already provided, try doing a restriction digest of your eluted product with some enzyme that would produce a recognizable pattern (I am assuming you have a map or sequence as a reference), then run that on a gel. It is possible that the big band near the top of your gel IS your plasmid, and that the fainter band down closer to your ladder is leftover sheared genomic. I have seen that happen in purifying large (90 kb) “wild type” resistance plasmids from clinical species, both by kit (I have used the same Qiagen kit) and old “kitchen sink” methods. When you digest a prep that has both plasmid (in abundance) and genomic (as a minor contaminant) and you run it on a gel, you get a nice pattern of a limited number of bands for the plasmid and a background of many faint bands for the genomic. But if the genomic is the one in abundance, you’ll have many faint bands and the plasmid bands are obscured. Also, I have been known to bring my gel concentration down to 0.5% for large uncut plasmids, though that makes the gels very hard to handle (if it is “standard” agarose).
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Re: BAC DNA purification

Postby 29yrsExperience » Mar 12 2018 6:17 pm

I found a gel photo that illustrates what I mean. This is a 0.8% agarose/TAE gel comparing two large 90-95 kb plasmids that have been digested as listed below. You can see the faint background bands from leftover genomic (E. coli DH5 alpha in this case). The important thing to know is that both plasmids have a single XbaI site, but while plasmid B has a single XhoI site, plasmid A has NO XhoI sites. So in sample lane 9, there is a chunk of uncut DNA riding much closer to the well.

The kit for these preps was Macherey-Nagel NucleoBond Xtra BAC, and it does not have an exonuclease treatment included. For my purposes the genomic background was not a hindrance.

Ladders: Invitrogen 1 kb plus ladder (top size is 12 kb)
Sample lanes:
1. Plasmid A, EcoRI; 2. Plasmid B, EcoRI, 3. Plasmid A, EcoRI-XbaI; 4. Plasmid B, EcoRI-XbaI; 5. Plasmid A, XbaI; 6. Plasmid B, XbaI; 7. Plasmid A, EcoRI-XhoI; 8. Plasmid B, EcoRI-XhoI; 9. Plasmid A, XhoI; 10. Plasmid B, XhoI
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Re: BAC DNA purification

Postby kiruphagaran » Mar 14 2018 4:35 pm

Thank you very much for the suggestions. I tried digesting it with many enzymes and concentrations but still I see the band near the well. I also increased incubation time after exonuclease addition but the band above 10 kb disappears but still the band near the well is present. In your experience after adding the EX buffer did the pellet dissolve completely? In my case I see a undissolved clump after adding buffer EX.

Thanks,
kirupa
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Re: BAC DNA purification

Postby 29yrsExperience » Mar 27 2018 5:05 pm

Checking my notes for an actual BAC prep I did with Qiagen Large construct kit in 2005, I see I had a large pellet of DNA at step 10, prior to adding buffer EX. As far as I can tell, my large pellet must have gone into solution when I added the EX, as I made no notes otherwise. After step 17, the post-exonuclease precipitation, I had a “tiny” pellet. I resupended it in 100 ul TE, pH8, and left it at RT to dissolve over the weekend. My DNA conc. was then 397 ng/ul on an Eppendorf Biophotometer. When run on a 0.7% agarose gel, the uncut BAC DNA ran as a smear near the loading well, with only a little evidence of genomic farther down. Cut with XbaI, however, it showed many fine bands from about 15 kb on down (but not as many as if I’d digested a whole bacterial genome). I later successfully subcloned the fragment I wanted out of it. Sorry I don't have a good image of the photo. But I don’t think I had problems getting the pellet into the EX buffer, to answer your question.
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Re: BAC DNA purification

Postby kiruphagaran » Aug 01 2018 1:52 pm

Hi all,
This is regarding the BAC prep in my previous post. I am purifying 106 kb BAC DNA. I get 2 forms of BAC DNA ( I don't know whether it is supercoiled and circular single stranded or nicked plasmid). But iam unable to attach the gel pic here. Could someone help with it?

Thanks
kirupa
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Re: BAC DNA purification

Postby 29yrsExperience » Aug 01 2018 2:16 pm

Hi Kirupa,

I have not been able to upload a photo to this site in a while; tried yesterday, in fact. I suspect something is a little wrong with the website. For a few weeks I could not even log in. I tried reading the FAQ in the help section and got no useful answer. And it is hard to answer your kind of question without seeing your picture. All I can suggest is that if you cut the BAC with a restriction enzyme, you should see the distinctions disappear and you should get a neat pattern of bands. With such a large plasmid (BAC) it's hard to predict how the uncut forms will migrate, or even if they will resolve at all.

So, does anyone else know how to get the photo uploading function fixed? I know a lot about cloning, but diddley about websites.
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Re: BAC DNA purification

Postby kiruphagaran » Aug 01 2018 2:32 pm

Thank you very much for your suggestions - 29yrsExperience. I tried restriction digestion but not getting clear pattern. I hope website will be fine so that I can upload the pic.
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Re: BAC DNA purification

Postby 29yrsExperience » Aug 01 2018 5:05 pm

I guess after all this time I should ask what the purpose of this BAC is? Are you going to subclone something out of it? Use it as a PCR template? Sequence it? Transform it into another host? Pardon me if you answered this somewhere in this long conversation and I just missed it. Depending on what it is for, you might just move on to the next step and see what happens.

Come to think of it, a successful transformation with some of the BAC DNA prep (I assume it has a selectable marker) should indicate that it is in fact BAC DNA and (mostly) intact.
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Re: BAC DNA purification

Postby kiruphagaran » Aug 01 2018 5:50 pm

Hi this BAC DNA is for microinjection in to mouse embryo to produce a transgenic mice. Thats why I need a pure prep.
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