Nucleic%acid%purification%

2.6.1. Genomic DNA extraction

Method&adapted&by&myself&from&that&of&Dr.&Andrew&Bassett.&

C.&reinhardtii&cells&were&grown&in&standard&liquid&culture&conditions&in&TAP&

medium&until&midQlog&phase&was&reached&(when&cell&density&reaches&1Q5×10⁶&

cells/ml).&Cell&density&was&calculated&using&optical&measurements&(Section&2.3).&

50&ml&of&culture&was&centrifuged&at&800&×g&for&15&min&to&pellet&the&cells.&

The&pellet&was&resuspended&in&4&ml&of&the&lysis&buffer&(1%&bovine&serum&albumin&

(BSA),&1&mM&EDTA,&0.5&M&sodium&phosphate&(NaPO4)&pH&7.2,&and&7%&sodium&

doecyl&sulphate&(SDS))&and&40&μl&of&proteinase&K&was&added&to&the&solution.&The&

samples&were&incubated&overnight&at&55°C.&Then&5&ml&of&

phenol:chloroform:isoamyalcohol,&45:23:1&(Life&Technologies)&was&added&to&each&

sample&and&each&sample&gently&inverted&for&15&sec.&The&samples&were&

centrifuged&at&4500&×g&for&10&min&at&room&temperature&and&the&upper&phase&

transferred&to&a&new&1.5&ml&tube.&The&phenolQchloroform&extraction&step&was&

repeated&once&more.&

OneQfifth&volumes&of&5&M&sodium&chloride&(NaCl)&and&2.5&volumes&of&pure&

ethanol&was&added&and&the&samples&gently&inverted&for&10&sec.&The&sample&was&

incubated&at&4°C&for&30&min&before&the&sample&was&centrifuged&for&30&min&at&

13,000&×g&at&4°C.&The&pellet&was&washed&with&5&ml&of&70%&(v/v)&ethanol&and&was&

centrifuged&for&5&min&at&4500&×g&at&4°C&to&collect&DNA.&The&supernatant&was&

removed&and&the&pellet&was&airQdried.&The&pellet&was&then&resuspended&in&300&µl&

RNase&solution,&transferred&to&a&1.5&ml&Eppendorf&tube,&and&incubated&at&40ºC&

for&1&hour.&Then&300&µl&of&phenol:chloroform:isoamyl&alcohol,&(45:32:1,&Life&

Technologies)&was&added&to&the&sample&and&the&sample&was&centrifuged&for&3&

min&at&10,000&×g&at&room&temperature&and&the&upper&phase&was&transferred&to&a&

new&tube.&

DNA&was&precipitated&by&adding&one&tenth&volumes&of&3&M&sodium&acetate&

(NaOAc)&and&1&ml&pure&ethanol.&The&visible&DNA&precipitate&was&transferred&to&a&

new&tube&containing&1&ml&70%&^&(v/v)&ethanol.&The&sample&was&centrifuged&for&5&

min&at&20,000&×g&at&room&temperature&to&collect&the&DNA.&The&pellet&was&washed&

twice&using&1.5&ml&70%&(v/v)&ethanol&and&centrifuging&for&5&min&at&20,000&×g&

between&each&wash.&The&pellet&was&then&air&dried&and&resuspended&in&50&µl&of&

distilled&water.&

Concentration&and&purity&of&the&DNA&was&checked&using&the&Qubit&system&(0).&

Quality&of&DNA&was&also&checked&by&running&1&µl&on&a&2%&(w/v)&agarose&gel,&

stained&with&ethidium&bromide,&quantified&using&Hyperladder&I&(Bioline),&and&

visualised&on&a&long&range&UV&box&(DarkReader&Transilluminator,&Clare&Chemical&

Research).

2.6.2. RNA extraction

RNA&was&extracted&according&to&a&protocol&modified&from&Molnar,&Schwach,&

Studholme,&Thuenemann,&&&Baulcombe,&2007.&

C.&reinhardtii&cells&were&streaked&to&a&single&cell&density&onto&solid&minimal&

medium&and&grown&in&standard&solid&culture&conditions.&A&single&colony&was&used&

to&inoculate&50&ml&of&liquid&minimal&medium,&which&was&grown&in&standard&liquid&

culture&conditions&until&midQlog&phase&was&reached&(1Q5×10⁶&cells/ml).&Then&50&

ml&of&culture&was&centrifuged&at&800&×g&for&15&min&to&pellet&the&cells&and&the&

supernatant&removed.&The&pellet&was&frozen&in&liquid&N2&and&stored&at&Q80ºC.&

To&extract&the&RNA&from&the&frozen&tissue,&the&pellet&was&resuspended&in&6&ml&of&

TRIzol&reagent&(15596Q026,&Life&Technologies)&and&the&sample&was&kept&on&ice.&

The&polysaccharides&were&then&pelleted&by&centrifugation&for&15&min&at&4000&×g&

at&4°C.&The&upper&phase&was&transferred&to&a&fresh&15ml&tube&and&incubated&for&5&

min&at&room&temperature&to&ensure&the&complete&dissociation&of&nucleoprotein&

complexes.&Then&1.2&ml&of&chloroform&was&added&after&which&the&samples&were&

vortexed&for&15&sec&and&once&again&incubated&for&5&min&at&room&temperature.&

The&resulting&mixture&was&centrifuged&for&15&min&at&4000&×g&at&4°C&and&the&upper&

phase&was&transferred&to&a&fresh&15&ml&tube&and&kept&on&ice.&RNA&was&collected&

by&centrifuging&the&samples&for&30&min&at&4000&×g&at&4°C&and&the&supernatant&

removed&by&aspiration.&Residual&salts&were&then&removed&by&rinsing&the&pellet&

with&8ml&of&80%&ethanol&and&immediately&afterwards,&the&RNA&was&again&

collected&through&centrifugation&for&5min&at&4000&×g&at&4°C.&Once&the&

supernatant&was&removed&the&pellet&was&again&rinsed&with&8&ml&of&80%&ethanol&

and&centrifuged&for&5&min&at&4000&×g&at&4°C.&Special&care&was&taken&to&remove&all&

supernatant&and&the&pellet&was&airQdried&at&room&temperature&for&3Q5&min.&The&

samples&were&placed&on&ice&and&the&pellets&resuspended&in&100&µl&of&RNaseQfree&

water.&To&allow&the&RNA&to&rehydrate,&the&sample&was&incubated&for&15min&on&

ice&and&then&vortexed&for&15&sec.&

Concentration&and&purity&of&the&RNA&was&checked&(Appendix&2.7).&Quality&of&RNA&

was&also&checked&by&running&2&µl&on&a&10%&TBE&precast&gel&(456Q5013QMSDS,&BioQ Rad,&CA,&U.S.A.),&stained&with&SYBR&gold,&quantified&using&Hyperladder&I&(Bioline),&

and&visualised&on&a&long&range&UV&box&(DarkReader&Transilluminator,&Clare&

Chemical&Research).

2.6.3. Agarose gel extraction of nucleic acid

DNA&separated&by&agarose&gel&electrophoresis,&stained&with&ethidium&bromide&

and&visualised&on&a&long&range&UV&box&(DarkReader&Transilluminator,&Clare&

Chemical&Research),&was&excised&from&the&gel&using&a&sterile&straight&edge&razor.&

DNA&was&extracted&and&purified&using&the&QIAquick&Gel&Extraction&kit&(28706,&

Qiagen,&Limburg,&Netherlands).&