5 XCES

5x Combinatorial Enhancer Solution

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.

INGREDIENT1 mL vial
Total volume of media1 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)* DMSO67 µL
Distilled water192 µL

* Final concentration in the CES

Method for making 5xCES:

  1. 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.
  2. 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.
  3. Add 67 µL DMSO per 1 mL CES
  4. For 1 mL CES, dissolve 0.001 g DTT (smells bad, use fume hood) in 200 µL water, and add to the mix above.
  5. Add water up to 1 mL (should be 192 µL water).
  6. Filter sterilize using the nylon filters
  7. 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 Communications 347: 747–751.

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Original Recipe by: J. Gallie Date: 20 Sept 10

Recipe Updated by: ___ Date: ___

Categories: Recipes