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Low Yield Miniprep

DNA miniprep maxiprep

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#1 cytheon

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Posted 20 January 2015 - 07:06 PM

Hi everyone,

 

I'm having a problem with my miniprep yields. Usually I get 100-300 ng/uL with a 1.5 mL culture using the Qiagen kit. I do plasmid prep frequently, but recently I've been getting around 30-50 ng/uL all of a sudden. I've tried doing all the troubleshooting listed in the manual (extra wash step, incubate elution buffer, use more bug culture, etc.), using a brand new kit, fresh antibiotics (and testing multiple plasmids with different resistances), even using Macherey-Nagel's Nucleobond miniprep kit. Other users in my lab get the same results, so I don't think it's something I'm doing wrong technically.

 

My maxipreps, on the other hand, are great and I consistently get over 2 mg from a 500 mL culture (from the original miniprep culture that I can't seem to get plasmid from using a miniprep kit). I can't afford to maxiprep everything though, so I'm trying to figure out what's going on.

 

Any thoughts or suggestions would be greatly appreciated!



#2 CPRES

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Posted 20 January 2015 - 07:17 PM

If you think you have taken care of everything listed in the troubleshooting manual, the next step would be to give your colony (not liquid culture) to a friend in the same or different lab who always gets good yield. If she gets a reasonable yield (~300-500 ng/ul of a high copy number plasmid), you buy her a coffee and begin looking at everything all over again.

 

Troubleshooting together have made more matches in science than successful co-authorships. But I digress.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#3 Leishman001

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Posted 21 January 2015 - 07:35 AM

Hi:

 

I too have had occasional problems with plasmid preps.

One thing to watch out for is old wash buffer. The ethanol can evaporate over time, leaving the aqueous fraction behind. It smells like there is ethanol present, but you're essentially eluting away your DNA at the wash steps. You can make your own wash buffer with by mixing ethanol to 80% final concentration with 10mM Tris-Cl pH 8. The key component in the wash is the ethanol which helps keep the DNA on the column.

 

Good luck!



#4 CPRES

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Posted 21 January 2015 - 08:53 AM

Yup, that would do it.


So. Now that you have your first ever question on bioforum answered (or not), mail yourself your username and password so you don't forget them, and then come back soon to update us on how it all worked out. That's how you build Karma in science.


#5 Trof

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Posted 25 January 2015 - 03:19 PM

But using a brand new kit probably would fix the "old wash" problem..

 

If it's consistent over different people and plasmids, I would first look for something that is same for all, like.. the incubator. The thermostart my be faulty and keeps lower than optimal temperature or something. In that case all the bacteria would grow more slowly and got lower yield. But that probably would be visible on the culture tube, as a less thick medium. 

 

Probably you can try growing the culture for more time than usual, if you get better yield, the growth was suboptimal. If not, or worse, then the culture already hit the peak and there is some other problem.


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#6 labtastic

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Posted 27 January 2015 - 10:08 AM

Are the vectors the same from before and now? I not uncommonly get 30-80 ng/ul with pET vectors (and other plasmids with the same origin of replication).

 

I usually use 4-5 ml of overnight culture grown in 2xYT media. Gives a few more cells, and sometimes better yields.

 

I also do a 5 min spin to dry off the ethanol before elution...Qiagen recommends 1min but I've seen too many people with leftover ethanol in their eluted dna.

 

If all those check out, I should note that Qiagen is having serious issues with their mini-prep columns (not their PCR cleanup columns).  We received multiple (>3) kits in which the column packing material was disintegrating during centrifugation and coming out the bottom. Each time we asked Qiagen for a replacement, and to their credit they sent them out very quickly, but once we started receiving replacement kits as bad as the kit they were replacing we decided enough was enough. And a disproportionate number of sequencing reactions were failing, and those that worked were always much shorter reads than we should have been getting.

 

So we just went to a new company. Now all our minipreps are solid, no problem with sequencing, PCR's work great off the miniprepped plasmids, yields are reliable. We have removed all Qiagen kits from our lab.  sad.png


Edited by labtastic, 27 January 2015 - 10:09 AM.






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