RNA Detection

Incubate the plate overnight at 37C. Select a single colony, and culture in a liquid LB medium overnight at 37C, and isolate ...

Reaction progress can be monitored using the Kaiser test. The resin should be washed until the waste solution is colorless. We ...

by World Premier International Research Center Initiative (WPI), MEXT, Japan. This work was inspired by the international and in ...

Robertson DL, Joyce GF (1990) Selection in vitro of an RNA enzyme that specifically cleaves single-stranded DNA. Nature 344:467 ...

Chapter 21 RNA Live Imaging in the Model MicroorganismUstilago maydis Sabrina Zander, Kira M€untjes, and Michael Feldbr€ugge Abs ...

[8, 9]. Endosomal movement along microtubules is mediated by the concerted action of plus-end directed kinesin-3 type motor Kin3 ...

Rrm4-containing mRNPs with the endosomal surface by a lipid- binding FYVE domain (Fig.1a;[9, 18]). In essence, microtubule- depe ...

2 Materials 2.1 Plasmids 1. pmax pona 12xTRICK 24xMS2SL (Addgene #64542, [25]). Potef-mCherry-cdc3-16boxB-3^0 UTR (Addgene #864 ...

Nitrate minimal medium (NM): 0.3 w/v % KNO 3 , 6.25 v/v % salt solution, add deionized water, adjust pH with 5 M KOH to 7.0, ad ...

achieved by integration at the endogenous locus using standard techniques adapted from model fungi [32–34]. In addition, the cor ...

linearized constructs can be transformed intoU. maydisproto- plasts. A detailed visual protocol showing the procedure is availab ...

Transform the generated Rrm4-GFP expression plasmid into U. maydisas described in Subheading3.2 [32–34]. Inoculate yeast-like g ...

Fig. 2Components of the endosomal mRNA transport. (a) DIC image of an Rrm4-GFP expressing hypha 6 h after induction of hyphal gr ...

3.4 RNA Live Imaging of Endosomal mRNA Transport in U. maydis Shuttling mRNAs in hyphae ofU. maydiscan be also detected using th ...

Use the 63or the 100objective and use a laser power (100 mW) of 20–30%. Record movies with 150 frames and 150 ms exposure tim ...

3.6 RNA Live Imaging Comparing Different Heterologous RBPs In contrast to the already described boxB loops, the PP7 stem- loop ...

Cotransform the desired constructs inU. maydisas described in Subheading3.2 (seeNote 14). Perform the microscopic analysis as d ...

For quantification of the number of moving mRNAs analyze about 30 cells. Calculation of the number of moving mRNA per cell is d ...

For the generation of kymographs other software packages than MetaMorph like the Zeiss software (Zen; Zeiss) or Fiji (ImageJ,ht ...

References Buxbaum AR, Haimovich G, Singer RH (2015) In the right place at the right time: visualizing and understanding mRNA l ...