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tRNA Probes

Wheat Germ Translation Systems

To provide the researcher with maximum flexibility in designing experiments, our wheat germ extract is purified by gel filtration chromatography to remove all endogenous small molecules (amino acids,  nucleotides, etc.) and supplied with minimal salts.  This approach allows the researcher to optimize the translation of his/her mRNA for [Mg2+], [K+], amino acid content, reducing agents, and/or other small molecules in the presence of various amounts of wheat germ extract, SRP, microsomes, aa-tRNAs, and/or other macromolecules.

Wheat germ extract (WG) is usually prepared with dithiothreitol (DTT) as the reducing agent. However, some WG is prepared with glutathione as a reducing agent rather than dithiothreitol for use in photo-crosslinking experiments. The optimal amount of WG added to a 25-μl translation incubation varies from 2-6 μl, depending upon the mRNA and microsomes, if any, added. Each 600 μl purchase therefore typically provides 120-200 25-μl assays, but may provide from 100-300 25-μl assays.
 
Wheat  Germ  Translation  Kit
 
WG can be purchased complete (cpt)  with the desired mixture(s) of 18-20 amino acids and an energy generating system (EGS) that contains phosphocreatine, creatine phosphokinase, ATP, GTP, spermidine, S-adenosyl-methionine (SAM), and protease inhibitors (PIN).  Each cptWGG or cptWGD contains a total of 600 ul of wheat germ extract, split into three 200-ul aliquots, as well as five 100-μl tubes of 10x Translation Mix (TM) and three 100-μl tubes of the desired amino acid mix (see below).  Customers must specify which mixture(s) of amino acids they desire.

 

Item

Description

Quantity of WG Extract

Cost per 600 ul

cptWGD
WGD + Trans. Mix + AAs   600 μl $ 100
cptWGG WGG + Trans. Mix + AAs   600 μl   $ 100
 
Wheat  Germ  Extract  Only
 
Wheat germ extract is also available for purchase without amino acids or EGS.
 
Item

Description

Quantity of WG Extract

Cost per 600 ul

WGD
Wheat germ extract with 2 mM DTT 600 μl  $ 80
WGG Wheat germ extract with 2 mM glutathione 600 μl  $ 80

Translation Components

Item

Description

Quantity per tube

Cost per tube

TM
10x Translation Mix     100 μl    $ 14
AA
AA-C
AA-K
AA-M
AA-MC
AA-MK
Various  aa  Mixtures    100 μl   $ 6

 

When [35S]Met is added to a translation, no non-radioactive methionine is added to the translation (= –M).  When Lys-tRNALys or one of its derivatives is added to a translation, no lysine is added to the translation to minimize competition from endogenous Lys-tRNALys (= –K).  Similarly, when Cys-tRNACys or one of its derivatives is added to the translation, no cysteine is added to the translation to minimize competition from endogenous Cys-tRNACys (= –C).  Please specify exactly which three tubes of amino acid mix are desired from the choices below:


If both [35S]Met and an aa-tRNA are added to a translation, choose:


for Lys-tRNALys and derivatives           AA–MK
for Lys-tRNAamb and derivatives         AA–M
for Cys-tRNACys and derivatives          AA–MC

If only an aa-tRNA is added to the translation, choose:


for Lys-tRNALys and derivatives           AA–K
for Lys-tRNAamb and derivatives         AA          (= all 20 amino acids)
for Cys-tRNACys and derivatives          AA–C

If a Cys-tRNA and a Lys-tRNA are added to the same translation, choose:


for Cys-tRNACys, Lys-tRNALys and derivatives           AA–KC