Murine RNase InhibitorÊÇÔڴ󳦸˾úÖбí´ï´¿»¯µÄÖØ×éÊóÔ´RNaseÒÖÖÆ¼Á£¬ËüÒÔ1¡Ã1µÄ·Ç¹²¼Û½áºÏ·½Ê½ÌØÒìÐÔÒÖÖÆRNase£¨A¡¢BºÍC£©£¬´Ó¶ø±£»¤RNAÍêÕû¶È²»±»½µ½â¡£±¾²úÆ·¿ÉÓë¸÷ÖÖÉÌÒµ»¯Reverse TranscriptaseºÍDNA Polymerase¼æÈÝ£¬¿ÉÓÃÓÚºóÐøRT-PCRÊÔÑéµÈ¡£Murine RNase Inhibitor²»º¬ÈËÔ´µ°°×ÖÐÁ½¸ö¶ÔÑõ»¯·Ç³£Ãô¸ÐµÄ°ëë×°±ËᣬÏà±ÈÈËÔ´RNase inhibitor¾ßÓиü¸ßµÄ¿¹Ñõ»¯»îÐÔ£¬¸ü¼ÓÊʺÏÓÚ¸ßŨ¶ÈDTTÊÔÑ飨ÈçqPCR£©¡£
1.RT-PCR¡£
2.ÔÚÌåÍâת¼/·Òë(È粡¶¾ÌåÍâ¸´ÖÆÌåϵ)ÖжÔRNA½øÐб£»¤¡£
3. RNAÍêÕûÐÔ±£»¤ÊÔÑé¡£
ÒÖÖÆ5ng RNase A 50%»îÐÔËùÐèÒªµÄøÁ¿¶¨ÒåΪ1¸ö»îÐÔµ¥Î»(U)¡£
-20¡ãC±£´æ£¬Àä¶³ÔËÊä¡£
1. ÔÚRNase-freeÀëÐĹÜÖÐÅäÖÆÉÏÊö·´Ó¦Ìåϵ»ìºÏÒº£º
RNase-free ddH2O | to 20 ¦ÌL |
5 ¡Á HiScript Buffer | 4 ¦ÌL |
Oligo (dT)18 (50 ?M) | 1 ¦ÌL |
dNTP Mix (10 mM each) | 1 ¦ÌL |
Murine RNase Inhibitor (40 U/?l) | 1 ¦ÌL |
HiScript Reverse Transcriptase (200 U/?l) | 1 ¦ÌL |
Ä£°åRNA | 10 pg – 2.5 ¦Ìg |
2. ÇáÇá»ìÔÈ£»
3. 50¡æ 45 min£¬70¡æ 15 min£»
4. ²úÎïÓÚ-20¡æ±£´æ¡£
1. ±¾²úÆ·µÄ»îÐÔζÈÔÚ42¡æ±íÏÖ×î´ó»îÐÔ£¬50¡æÒÔÉÏʧ»î¡£
2. ±¾²úÆ·ÒÖÖÆ RNase »îÐ﵀ pH Öµ·¶Î§½Ï¹ã£¬ÔÚpH 7.0 – 8.0ʱ±íÏÖ×î´ó»îÐÔ¡£
3. ±¾²úÆ·ÔÚʹÓÃǰ±ÜÃâ¾çÁÒÕðµ´£¬Çᵯ»ìÔȽϺá£
4. ±¾²úÆ·²»ÒÖÖÆRNase H¼°RNase T1»îÐÔ¡£
ÎïÁ÷ƽ̨£ºÄ¬ÈÏΪ¾©¶«ÎïÁ÷¡£ÈôÓÐÆäËûÐèÇó£¬ÇëÁªÏµ¶Ô½ÓÏúÊÛ¡£
·¢»õʱ¼ä£ºÈ·È϶©µ¥²¢Íê³É¸¶¿îºó1~2¸ö¹¤×÷ÈÕÄÚ°²ÅÅ·¢»õ¡£
·¢»õ·½Ê½£º±ù´ü»ò¸É±ù¡£