The applicants can use their own constructs for the following practicals:
- Methyl specific labelling of proteins
- 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:
- Methyl specific labelling of proteins:
The plasmid used for the protein expression must be kanamycin-resistant (pET-41 or pET-28 preferred) and the construct should have an His-tag. The protein should be expressed in the soluble fraction in M9 minimal media (prepared in H2O) with a yield higher than 10 mg/L. For logistical reasons, we are limited to using E. coli BL21(DE3) during the Practical School and all protein expression will be conducted overnight at 20°C.
- 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 February 15th 2019.
Constructions received after February 15th 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.