Paul.xu at 03:32, 22 June 2017

2017-06-22T03:32:51Z

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== 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.''

{| border="1"
|-
! align="right" | INGREDIENT
! align="right" | 1 mL vial
|-
! align="center" | Total volume of media
! align="right" | 1 mL
|-
| align="center" | 2.7 M* betaine (B2629-50G Sigma, MW 117.15)
| align="center" | 540 µL
|-
| align="center" | 6.7 mM* didithiothreitol (DTT, Cleland's reagent) (43819-1G Sigma, MW 154.25)
| align="center" | 200 µL
|-
| align="center" | 55 ug/mL* BSA (albumin bovine serum fraction, A3059-50G from Sigma)
| align="center" | 1 µL
|-
| align="center" | 6.7% (v/v)* DMSO
| align="center" | 67 µL
|-
| align="center" | Distilled water
| align="center" | 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 Communications'''''347:''' 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 ==

2-O-b-Mannosylglycerate,

trehalose

glycerol

ectoine

diglycerol phosphate

Rhodafac RE-960 POLYOXYETHYLENE LAURYL ETHER PHOSPHATE https://www.google.com/patents/EP2152864A1

Enzyme stabilizer - Revision history

Paul.xu at 03:32, 22 June 2017