LAB Media: Difference between revisions
(→Notes) |
(→Notes) |
||
| Line 46: | Line 46: | ||
This media was derived from the work of Brody JR and Kern SE. One of the major problems with [[SB]] media is compression of the higher molecular weight bands. In Singhal H, Ren YR, Kern SE they found that you can alleviate these problems in Lithium Borate media by having the pH near 7. However, their protocol makes it difficult to make a stock solution (they take lithium hydroxide and pH with boric acid - to get the 10mM lithium hydroxide to pH 7 requires near saturating amounts of boric acid). Starting with lithium acetate instead of lithium hydroxide means that the final pH should be near 7 after the addition of 10mM boric acid. | This media was derived from the work of Brody JR and Kern SE. One of the major problems with [[SB]] media is compression of the higher molecular weight bands. In Singhal H, Ren YR, Kern SE they found that you can alleviate these problems in Lithium Borate media by having the pH near 7. However, their protocol makes it difficult to make a stock solution (they take lithium hydroxide and pH with boric acid - to get the 10mM lithium hydroxide to pH 7 requires near saturating amounts of boric acid). Starting with lithium acetate instead of lithium hydroxide means that the final pH should be near 7 after the addition of 10mM boric acid. | ||
Since lithium acetate, however, is about 6-fold more expensive compared to lithium hydroxide, you can prepare < | Since lithium acetate, however, is about 6-fold more expensive compared to lithium hydroxide, you can prepare <q>cheap</q> lithium acetate by using equimolar amounts of acetic acid and lithium hydroxide. There are two caveats with this procedure. (1) First solve the lithium hydroxide, subsequently add the boric acid, and eventually add acetic acid. Mixing the components in this order will avoid heating of the solution as it will happen when you mix the hydroxide and acetic acid first. Additionally, the boric acid will be resolved much faster applying this order. (2) Check pH after mixing the components. A pH around 6.5 in the 50x stock will result in pH 7.5 in the final solution (which you should not exceed). | ||
This media can be reused many times if it is rebottled to prevent evaporation. | This media can be reused many times if it is rebottled to prevent evaporation. | ||
Revision as of 05:55, 11 September 2013
Curators
Abstract
Lithium Acetate Borate (LAB) buffer is an agarose gel electrophoresis media for DNA gels. It has low conductivity and allows for less heat buildup and thus higher voltage and faster runs. Compared to TAE and TBE, gels can run faster in this media and this media is easier to prepare and costs less. Compared to SB you will be able to run a little faster and you will have less compression of the higher molecular weight bands and you can easily make a 25x stock and possibly a 50x stock. This media is compatible with commercial gel extraction kits.
Materials
- Lithium acetate dihydrate - Sigma-Aldrich L6883 - Molecular Weight 102.02
- Boric acid - Sigma-Aldrich B7901 - Molecular Weight 61.83
Procedure
1x Solution:
- 10mM Lithium Acetate
- 10mM Boric Acid
For a 1L 1x Solution:
- 1g Lithium acetate
- 0.62g Boric acid
- make up 1L with dH2O
25x Solution:
- 250mM Lithium acetate dihydrate
- 250mM Boric acid
For a 1L 25x Solution
- 25.5g Lithium acetate dihydrate
- 15.5g Boric acid
- make up 1L with dH2O
Dilute to 1x and use it to make the gel and to run the gel. Do not mix buffer systems.
Ethidium bromide can be added to the gel.
Run the gel at 15-20 V/cm (possibly higher) for 15-20 minutes (it's good to set a timer).
Notes
This media was derived from the work of Brody JR and Kern SE. One of the major problems with SB media is compression of the higher molecular weight bands. In Singhal H, Ren YR, Kern SE they found that you can alleviate these problems in Lithium Borate media by having the pH near 7. However, their protocol makes it difficult to make a stock solution (they take lithium hydroxide and pH with boric acid - to get the 10mM lithium hydroxide to pH 7 requires near saturating amounts of boric acid). Starting with lithium acetate instead of lithium hydroxide means that the final pH should be near 7 after the addition of 10mM boric acid.
Since lithium acetate, however, is about 6-fold more expensive compared to lithium hydroxide, you can prepare cheap
lithium acetate by using equimolar amounts of acetic acid and lithium hydroxide. There are two caveats with this procedure. (1) First solve the lithium hydroxide, subsequently add the boric acid, and eventually add acetic acid. Mixing the components in this order will avoid heating of the solution as it will happen when you mix the hydroxide and acetic acid first. Additionally, the boric acid will be resolved much faster applying this order. (2) Check pH after mixing the components. A pH around 6.5 in the 50x stock will result in pH 7.5 in the final solution (which you should not exceed).
This media can be reused many times if it is rebottled to prevent evaporation.
References
<biblio>
- Paper1 pmid=14989083
- Paper2 pmid=15351274
- Paper3 pmid=20593002
Specific Protocols
Discussion
You can discuss this protocol.
Tag this page with categories to allow easier indexing and searching. See Categories for information on existing categories.
