RNA prep from Chlamydomonas
1x10^8 cell 준비(cell 상태가 중요함)
seed culture 후 2-3일 배양한 cell 사용
1. Take the tube out of the liquid nitrogen and immediately add 1ml of Trizol to the frozen pellet colse the tube and vortex for 10min (Ensure that there is complete mixing). Incubate the homogenized sample for another 5min at room temperature to permit the complete dissociation of nucleoproitein complexes. Centrifuge the homogenized samples at 12,000 x g for 10min at 4℃ to remove insoluble material. [Note: Make sure that the centrifuge speed is correct using the g values(i.e, RCF not RPM).]
언상태의 cell 곧바로 add 1ml Trizol(언상태로 해야함 아님 깨짐) vortexer로 약간 풀어주고 for 10min, RT 5min, 12000g for 10min at 4℃
2. Transfer supernatant to a clean tube. Add 0.25ml of chloroform. Cap sample tube, shake vigorously by hand for 30seconds, and then vortex for 2min (Note: Make sure that the aqueous and organic layers are mixing, i.e., no visible clear layer at the bottom). Incubate the tubes at RT for another 2min and centrifuge at 12,000 x g for 15min at 4℃.
상등액 new tube에 1ml정도만(찌꺼기 조심) add 0.25ml chloroform. 손으로 흔듬 점점 세게 30s 후 vortex for 2min, RT 2min, 12000g for 10min at 4℃
3. Transfer the aqueous phase to a clean tube (approximately 55~60% of the volum of Trizol). Avoid the lower phenol-chloroform phase and the interphase. Add an equal volume of phenol/chloroform(1:1), vortex for 2min, then centrifuge as above.
상등액 new tube로(찌꺼지 조심) add 동일vol phenol:chloroform(IAA) pH6.6/7.9 Ambion
2번 step과 동일 x 2반복
9. Transfer the aqueous phase to a clean (RNase-free) tube. Add one volumn of isopropanol. Incubate on ice for 30~45min and precipitate the RNA by centrifugation at 12,000 x g for 20min at 4℃. Carefully remove the supernatant since the RNA pellet may not adhere tightly to the walls of the tube.
상등액 new tube로(찌꺼기 조심) add 동일vol isopropanol(RNA용 보관 침전시킴) 섞어줌30-45min or O/N 여기서 쉴수 있음. 12000g for 20min at 4℃
10. Wash the RNA pellet with 1ml of 75% EtOH (not 70%). Mix the sample by vortexing for 15~20min at room temperature. The pellet should become loose and break into smaller fragments. Centrifuge at 12,000 x g for 8min at 4℃. Carefully remove the supernatant without disturbing the loose pellet. Repeat the 75% EtOH wash once.
75% DEPC EtOH 미리 만들어 냉동고에 넣어둠. 1ml 손으로 살짝 inverting, 12000g for 8min at 4℃. x2반복. 1ml tip 딴후 10ul tip으로 다시 한번 더 땀
11. Air dry the pellet for 10~20min. Do not over-dry as this will decrease its solubility. Resuspend the pellet in 100ul of RNase-free TE buffer containing 1% SDS by passing the solution a few times through an RNase-free pipet tip. Note that up to 50% of the RNA may pellet as a smear on the wall of the tube. To recover the RNA it is necessary to wash the appropriate quaderant of wall multiple times. However, be careful to avoid losing pellet fragments that my stick to the pipet tip.
휴지 위에 뒤집어 엎어줌.
pellet이 안 녹을시 water bath 55℃ 5분 정도.
또는 DEPC 넣고 얼린 후 다음날에 nanodrop 확인 & gel loading 으로 RNA양 확인(TBE gel 만들어 바로 사용)
https://www.chlamycollection.org/methods/craigs-chlamy-corner/rna-prep/
RNA Prep - Chlamydomonas Resource Center
Source Aksoy, GrossmanBaierBarjonaBaulcombe, ValliBeck, Boynton-GillhamBoynton-GillhamCardineauCrozetDinnenyFerrisGarcía, CardineauGoldschmidt-Clermont, Boynton-GillhamHegemannHeitzerHerrinHoffman, Boynton-GillhamJiang, SternJohnsonJonikasKawabeKindle, Bo
www.chlamycollection.org
