The applicants can use their own constructs for the following practicals:
- Cell-free expression and specific labelling of soluble proteins
- In vitro production of specifically labelled RNA
The Organisers need to evaluate the feasibility of using an applicant's constructs prior the start of the Practical School.
Applicants wishing to use their own targets must ensure it meets the follow specifications:
- Cell-free expression and specific labelling of soluble proteins:
The target cDNA must be cloned into either pIVEX 2.4d or pEXT5-NT/TOPO with an N-terminal His-tag. We will need pure DNA, without RNA or RNAse contamination. Please send at least 60 ug of cDNA per construct at a concentration of 1 ug/uL in water.
- In vitro production of RNAs:
The transcribed target RNA sequence needs to start with at least 2 G (i.e. transcribed RNA sequence: 5' - GGNNNN...NNN-3') and the DNA matrix (double-stranded DNA oligonucleotides or plasmid) needs to contain a T7 promotor before the target RNA sequence (T7 promotor : 5'-TAATACGACTCACTATAGGNNNN...NNN-3'; DNA matrix: 5' - YYY....YYYYCCTATAGTGAGTCGTATTA -3', Y: complementary sequence of N). dsDNA oligonucleotides should be purified by reverse phase HPLC. Plasmid should be linearized and purified by users at the highest concentration possible in water. Send us 200 ug of "target" DNA matrix at a concentration of 100 uM in water
All constructs should be received by May 1st 2024.
Constructions received after May 1st or received on time but without respecting the above specifications will not be considered for the practical school. A range of model proteins and RNA systems will be available for use during the Practical School.