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Miniprep OD beyond 2.00 - Troubleshooting-help desperately needed


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10 replies to this topic

#1 greenhorn

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Posted 12 August 2011 - 07:17 AM

Hey everyone,

I am obtaining constantly 260/280 ratios beyond 2.00 (2.00-2.10) for my minipreps (dissolved in TE) and there is also a problem with sequencing of the constructs. When I cleaned those preps on columns, I lost over 90% of the yield (which was then not enough rest for sequencing). I made all solutions for the protocol new, but now I have the same problem again.

Does anyone have an idea what could be causing that problem and if there is any way to save my preps without losing everything?

Thank you very much!!

#2 greenhorn

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Posted 12 August 2011 - 07:54 AM

PS: The protocol I am using consists of 3 home-made solutions
1. GTE (50mM Glucose, 25mM Tris pH 8, 10mM EDTA) + RNAse freshly added = "solution buffer" (100ul/pellet) - suspend
2. "lysis buffer": freshly prepared NaOH-SDS (1%SDS, 200mM NaOH) (200ul/sample) - mix by inverting several times
3. "precipitation buffer" : 3M potassium acetate, 11.5% glacial acetic acid (150ul/sample) - mix immediately by inverting
put on ice 15min
spin high speed 15min 4C
transfer supernatant in new tube
add twice volume of EtOH 100%
spin high speed 20min 4C
discard supernatant, wash in 70% EtOH

#3 ranvi

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Posted 12 August 2011 - 08:05 AM

For Mini prep use Qiagen mini prep (100$ for 50 column) which always gives very good sequence results Dont need to clean up. If you want to clean up use Zymo research Dna concentrator which gives high yield in a small volume

#4 greenhorn

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Posted 12 August 2011 - 10:07 AM

Thank you very much!

But I am afraid the fact that I COULD HAVE used some commercial kit which probably WOULD HAVE resulted in proper values or otherwise I COULD blame the manufacturer doesn't really help me since it's all conjunctive and my preps are already isolated ;o)

#5 leelee

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Posted 13 August 2011 - 04:03 AM

I think that what ranvi means is that you could just redo the minipreps using a kit. And I agree, I would. It really isn't that much work to do it again.

If you really really want to use what you already have, you could try doing a phenol/chloroform extraction?

#6 leelee

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Posted 13 August 2011 - 04:04 AM

Also, which columns did you use for your clean up? It seems pretty astounding to lose 90% of your yield!

#7 phage434

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Posted 13 August 2011 - 08:06 AM

You almost certainly have large amounts of RNA in your prep, leading to high 260/280 ratios. You can probably get rid of it with RNAse A treatment, but these preps are pretty dirty, and may not be easy to clean up well enough for sequencing.

#8 greenhorn

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Posted 15 August 2011 - 11:57 AM

Hello everyone,

thank you very much for all your answers! I generally agree with the kit, but I am on a fellowship in another lab, most people are on holiday (including the ones who could order stuff) and I have 6 weeks left to finish my cloning and transfect the constructs...so I need to find the quickest possible solution (waiting to order, order and wait for delivery would cost at least 2 weeks assuming I could convince that this is the only solution...).
I meanwhile also tried the phenol-chloroform extraction, the improvement of the ratio was very poor (changed maybe from 2.05 to 1.99) and the loss of yield was about 80%. Maybe the RNAse I've been using is not good any more - I will try to exchange it, too. But shouldn't I get rid of RNA with the phenol/chloroform? And a colleague did the protocol without any problems not using any RNAse at all...?
I thought maybe it is some SDS/... strongly bound to my DNA...? Do you think that would be possible and do you have any idea how one could get rid of that? If possible without any commercial kits because that is an option I currently just don't really have...but I have almost any non-kit chemical etc. available... :o)

#9 ochiesan

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Posted 18 August 2011 - 08:04 AM

Hi everyone,
I have similar question with greenhorn, regarding the phenol-chloroform extraction.

I need to purify my DNA sample (plasmid, from E. coli) from RNA for making as a probe. I use the alkaline lysis procedure. The addition of RNase in the solution I did not remove all the RNA. So i did the additional RNase treatment after the first precipitation.

Though I can see the DNA pellet from the first precipitation (isolation from 100ml of culture), I often couldn't see any white pellet after phenol-chloroform extraction for removing RNase.

Will it be better if I use only chloroform instead of phenol-chloroform for extraction?

Thank you for any suggestion. And sorry if I'm a little off topic :")

#10 phage434

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Posted 18 August 2011 - 02:47 PM

A phenol/chloroform extraction should always be followed by a chloroform only extraction to remove excess phenol, which can totally inhibit downstream enzyme activity.

#11 ochiesan

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Posted 18 August 2011 - 07:33 PM

Thank you so much, phage434. Will try to isolate the DNA again.




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