in vitro translation:

General remarks:

Small amount of proteins can be synthesised by translation efficient cell lysates, like from reticulo cytes or wheat germ.Since the synthesised amounts are below the detection limit of coomassie stainin, radioactive Met is added to label the proteins.
The template is either mRNA ready for translation (with a 5' cap and a good 3'UTR) in the classical assay or a plasmid with the cDNA of choice under the control of a SP6, T7 or T3 promoter in the coupled transcription translation assay (TNT kit).Since you are working with RNA the reactions are sensitive to RNA degradation. You may wear gloves, use RNAase free tips and be concerned about possible RNAase contamination in general.
Set up the labelling reaction in the radioactivity room, use screw caps with a rubber seal to prevent leaking of radioaktivity.
The lysate is very sensitive and must not be repeatedly thawn and frozen. When opening a new 200 µl aliquot, distribute the remaining lysate in vials a 25 µl and freeze them immediately in liquid nitrogen.

coupled in vitro transcription and translation (TNT kit, Promega)

expression of proteins directly from a plasmid with an RNA polymerase site 5' to the cDNA
set up the labelling reaction in the radioactivity room, use screw caps with a rubber seal to prevent leaking of radioaktivity. Thaw the reagents and keep them on ice. Make sure that the buffers are completely dissolved. Set up the reaction on ice.

reaction mix (total 25 µl)

y µl water (DEPC treated)

x µl plasmid (0,5 - 1 µg)

1 µl reaction buffer

0,5 µl amino acid mix without methionine

0,5 µl RNasin (40 u|µl)

0,5 µl RNA polymerase SP6/T7/T3 (??? units)

12,5 µl retic lysate

1 µl [35S] methionine (> 1000 Ci/mmol, 10 µCi/µl)

mix and incubate for 90 min at 30°C
freeze in liquid nitrogen and store at -80°C (or-20°C)

with reticulocyte lysate (Amersham)

reaction mix: total volume 25 µl

12,5 µl retic lysate

0,5 µl amino acid mix (minus met)

1 µl Met[35S],

0,5 µl RNasin (40 u)

0.5 - 1 µg mRNA

X µl DEPC H2O to the total volume of 25 µl

The reacction mixture may be complemented by KCl and Mg-acetat to increase the efficiancy of translation.

- prepare reaction mix on ice
- incubate at 30ºC for 1-3 h
- store at -20ºC
- use 1 to 5 µl for the experiment
- SDS PAGE
- fix gel in 20% methanol, 10% trichlor acetic acid (TCA) for 30 min
- wash with water, to increase the pH
- incubate 30 min in 1 M Na-salicylat
- dry the gel
- expose a X-ray film

y µl	water (DEPC treated)
x µl	plasmid (0,5 - 1 µg)
1 µl	reaction buffer
0,5 µl	amino acid mix without methionine
0,5 µl	RNasin (40 u\|µl)
0,5 µl	RNA polymerase SP6/T7/T3 (??? units)
12,5 µl	retic lysate
1 µl	[35S] methionine (> 1000 Ci/mmol, 10 µCi/µl)