Use this category for questions directly related to DNA manipulation (isolation, purification, sequencing, etc.) and questions regarding general PCR methodologies
Moderators: r.rosati, mchlbrmn
We have some 10X TBE that unfortunately precipitated out of soln. The manufacturer (Amresco) said we could autoclave it, use NaOH to rehomogenize the material, stir while heating, or filter it (filter paper). I did the last two procedures and still had precipitate remaining. The tech rep was not very helpful in that she didn't know what concentration of NaOH we should use to rehomogenize the precipitate (that's my first question). My second question: is the the filtered 10X TBE soln I now have (at a pH of 8.33) even usable, and will the pH drop significantly with the addition of dH2O to make a soln of 1X TBE, or should I attempt to rehomogenize the precipitate with NaOH (is there anything else I could use?) and then add it back to the TBE soln? Amresco says this is a problem they are aware of (FYI, the expiration date of the TBE is 5/06).
- Posts: 3
- Joined: Jun 20 2004 11:08 pm
any TBE solution will precipitate over time. To get the stuff back into solution, you will probably just need to stir while heating
- Posts: 33
- Joined: Jun 09 2004 6:09 pm
Precipitation of TBE frequently occurs, especially when a 10x stock is prepared. 5x stocks are more stable according to Maniatis. Autoclaving will retard this reaction. Alternatively, filtration shortly after preparation through a 0.2-0.45 µm cellulose acetate or cellulose nitrate membrane will reportedly allow a 20x stock to be prepared. 30x stocks prepared by this method are stable for 2 weeks. Filtration suggests that nucleation of salt crystals by dust particles or other microscopic insoluble material is responsible for the precipitation problem. If turbidity develops in filtered buffer stocks, autoclaving for 5 minutes at 110°C will remove cloudiness.
- Forum Emeritus
- Posts: 501
- Joined: Oct 24 2002 1:42 pm
- Location: Boulder, CO
We usually use the microwave to remove any precipitate formed in our 10xTBE. This is fast and we never had any trouble with this.
Hope this helps...
- Posts: 7
- Joined: Apr 28 2003 10:50 am
- Location: Germany
An alternative is to prepare only what you need: you can make 1 l of 10X TBE and dilute it straight away to make up a 10 l tank of 1X TBE or scale down/up depending on your consumption.
- Prolific Post-Master
- Posts: 143
- Joined: Sep 17 2003 9:05 am
- Location: Scotland
As a side note, I've never had any problems using a stock that had precipitate in it - assuming there isn't a one inch precipitate at the bottom. Unless you need exact %s for some reason, I would go ahead and just use the 10x TBE and dilute it as is.
- Prolific Post-Master
- Posts: 112
- Joined: Jun 17 2004 7:14 pm
Buying TBE?! Such luxury. Its cheap and easy to make.
However you should find heating via microwave and stiring frequently should help.
If you filtered your solution with the precipitate, then you would have lost much of what makes TBE. If you make your own using ultrapure dH2O helps prevent this problem.
- Posts: 12
- Joined: Jun 02 2004 5:03 am
- Location: London, England
Return to DNA and General PCR Methods
Who is online
Users browsing this forum: r.rosati and 5 guests