Enzyme stabilizer
5x Combinatorial Enhancer Solution
[edit]Combinational Enhancer Solution (CES) used to improve the efficiency of PCR. Add 5 ul for 25 ul PCR reaction and 10 ul for 50 ul PCR reaction.
| INGREDIENT | 1 mL vial |
|---|---|
| Total volume of media | 1 mL |
| 2.7 M* betaine (B2629-50G Sigma, MW 117.15) | 540 µL |
| 6.7 mM* didithiothreitol (DTT, Cleland's reagent) (43819-1G Sigma, MW 154.25) | 200 µL |
| 55 ug/mL* BSA (albumin bovine serum fraction, A3059-50G from Sigma) | 1 µL |
| 6.7% (v/v)* DMSO | 67 µL |
| Distilled water | 192 µL |
* Final concentration in the CES
Method for making 5xCES:
- Make a 5 M stock of betaine, autoclave (it will dissolve properly only after autoclaving) and store at 4°C. Use 540 µL of this stock solution per mL of 5xCES required. This is kept as a stock solution in the fridge.
- Make a 55 mg/mL stock of BSA (0.055 g in 1 mL water, filter sterilize, store at 4'C). Add 1 µL of the stock solution per 1 mL 5xCES required. This is kept as a stock solution in the fridge.
- Add 67 µL DMSO per 1 mL CES
- For 1 mL CES, dissolve 0.001 g DTT (smells bad, use fume hood) in 200 µL water, and add to the mix above.
- Add water up to 1 mL (should be 192 µL water).
- Filter sterilize using the nylon filters
- Aliquot into 200 µL portions, store at -20°C (keeps for ~3 months)
Reference: Ralser, M., R. Querfurth, H. Warnatz, H. Lehrach, M. Yaspo, S. Krobitsch. 2006. An efficient and economic enhancer mix for PCR. Biochemical and Biophysical Research Communications347: 747–751
PCR Buffer: 65 mM Tris–Cl, 16.6 mM (NH4)2SO4, 3.1 mM MgCl2, and 0.01% (v/v) Tween 20, pH 8.8
List of known enzyme stabilizers
[edit]2-O-b-Mannosylglycerate,
trehalose
glycerol
ectoine
diglycerol phosphate
Rhodafac RE-960 POLYOXYETHYLENE LAURYL ETHER PHOSPHATE https://www.google.com/patents/EP2152864A1