In Vitro transcription

Talen mRNA preparation(20140225 更新版)

Plasmid digestion(不同的载体需要用不同酶切):

对于pCDNA6（单酶切进的载体）：

PVU2单酶切

Golden Gate 载体：

用Sca1 Sac1 Smal1三酶切。

后用酚氯仿异戊醇抽提，吸上清，加入二倍体积无水乙醇（RNase free）沉淀，用75%乙醇（DEPC-H2O稀释）洗一次，加10 ul DEPC-H2O溶解。

跑胶定量。

In vitro transcription:（AmpliCap-Max? T7 High Yield Message Maker Kit, CellScript, C-ACM04037）

体系：

Plasmid DNA Template：1 ug

5XTranscription Buffer：2 ul

ARCA Cap/NTP PreMix: 3 ul

RNA inbibitor: 0.5 ul

T7 Enzyme: 1 ul

Total reaction volume: 10 ul;

Incubate at 37 degree centigrade 1~2h

Add 1 ul DNase, 37 degree centigrade, 30min.

At the end of the DNase digestion, you may immediately proceed to the poly(A) tailing reaction or store the reaction at -20 degree centigrade overnight before proceeding to poly(A) tailing.

（留0.5ul，最后跑胶检测）

In vitro Poly(A) Tailing with Poly(A) Polymerase Tailing Kit.（A-Plus? Poly(A) Polymerase Tailing Kit, CellScript, C-PAP5104H）

体系：（50ul total）

10X reaction buffer：5 ul

Rnase Inhibitor：1 ul

Poly(A) Polymerase: 1 ul

10mM ATP: 5 ul

Invitro transcription reaction: 10.5 ul

DEPC-ddw: 27.5 ul

Incubate the reaction at 37 degree for 1h.

At the end of the the poly(A) tailing reaction, you may immediately proceed to RNA purification or store the reaction at -20 degree overnight before purification.

mRNA Purification with MEGAClear Kit:（MEGAclear? Kit（20 reactions），Ambion，AM1908）

Firstly, bring the RNA sample to 100 μL with Elution Solution. Mix gently but thoroughly.

i. Add the reaction components into a clean1.5-ml microcentrifuge tube at roomtemperature according to the order givenbelow:

ii. Add350μl of Binding Solution Concentrateto each poly(A) tailing reaction andmix gently by pipetting.

iii. Add250 μl of 100% ethanol and mix gently by pipetting.

iv. Add the RNA sample into thesame Filter Cartridge and centrifuge at10000 xg for 30 seconds. Decant the flowthroughliquid and return the Filter Cartridgeto the Collection Tube.

v. Add500μl of alcohol diluted Wash Solutionto each Filter Cartridge and centrifuge at 10000xg for 30 seconds. Decant theflow-through liquid and return the FilterCartridge to the Collection Tube. vi. Add another 500μl of alcohol diluted WashSolution to each Filter Cartridge andcentrifuge at 10000 xg for 30 seconds.Decant the flow-through liquid and return theFilter Cartridge to the Collection Tube.

ix. Dry the Filter Cartridge by centrifugation at10000 xg for 1minute.

x. Transfer the Filter Cartridge into a newCollection Tube. Add 50 μl of ElutionSolution directly onto the center of the filterinside the Filter Cartridge. Close the cap ofthe tube immediately.

xi. Heat the tube at 65 ?C for 10 minutes on aclean heat block or in a clean water bath.

xii. Centrifuge the tube at 16000xg for 1 minute to elute RNA.

测浓度，跑胶定量。