PS - SELEX

Overview to the project

Preparation of the DNA templates for in vitro transcription

done - XXXX clone names, location, protocol

In vitro transcription system - and for what we use the RNA

done - andreas

How much RNA required for PS project: 10 templates, more than 10 ug each

In summary a mixing of equimolar amount - about 100 ug or more.

How much RNA required for establishing the binding conditions of Nucleocapsid to RNA in EMSA

Use 1-3500 RNA - refolded - fragmented (100nt in length) - non-labeled. Native PAA gels and staining with SybrGold/Green.

Questions to solve: can we separate the NP-RNA complex from free RNA on the gel

RNA amount per lane: (2ug finally) for establishement 0.5ug. We require about 100ug to start with.

How much RNA required for LLPS project.

RNA 1-3500; and another control fragment from region 13000 or 20000.

per LLPS reaxn (10ul volume; 2uM protein; 5nM RNA) about 60ng RNA are required (pmid: 32587965; 5nM)

Conclusion: we can use it to compare and testing, but for screening it is to much material we need

make 100 ug for LLPS comaprative analysis.

How much RNA required for NP-RNA assembly (chromatin like assembly reactions + MNase)

I would think 100 ug are good for a start

Re-folding of the long RNA before fragmentation

Ongoing Valerie Wenzel

How to refold: see (32587965) see method: Annealing/Folding RNA - Method

Fragmentation of the RNA

Ongoing Valerie Wenzel

Shearing with bioruptor. Try to shear down to 100bp.

Analyse RNA-Fragment size with Tape-Station RNA tapes.

Maybe we should have a look at the Covaris - 5ug RNA works well per tube: dna-rna-shearing-for-ngs

Side Projects

• Establishment of RNA-Nucleocapsid binding in EMSA. see above
• RNA synthesis for the LLPS project. The folded RNA 1-3500 should be working better in LLPS (32587965). see above
• RNA synthesis for NP-RNA assembly. see above