±±¾©Ê¯ÓÍ»¯¹¤Ñ§Ôº2026ÄêÑо¿ÉúÕÐÉú½ÓÊÕµ÷¼Á¹«¸æ
²é¿´: 1099  |  »Ø¸´: 2

shaojing690

гæ (³õÈëÎÄ̳)

[½»Á÷] ¡¾·ÖÏí¡¿·´ÏòPCRµÄ²½Öè ÒÑÓÐ2È˲ÎÓë

·´ÏòPCR £¨inverse-PCR£©ÊµÑé²½Öè
inverse-PCRÊǿˡ²åÈëÆ¬¶Î²àÒíÐòÁзdz£ÓÐЧµÄ·½·¨¡£Í¨³£²ÉÓõÄTail-PCR¼ÙÑôÐÔÌ«¶à£¬¶øInverse-PCRÒ»°ãÖ»ÒªÓÐÌØÒìÌõ´ø£¬»ù±¾ÉϾÍÊÇÄ¿µÄƬ¶Î¡£
»ù±¾²½ÖèÈçÏ£º
1¡¢·´ÏòPCR£¨Inverse PCR£©Ô­Àí£º·´ÏòPCRÊǿˡT-DNA²åÈëλµã²àÒíDNAÐòÁзdz£ÓÐЧµÄ·½·¨¡£
a. Ñ¡ÔñºÏÊʵÄÏÞÖÆÐÔÄÚÇÐøλµã¡£²¢ÔÚT-DNAµÄ±ß½ç£¨×ó±ß½ç¡¢Óұ߽ç¾ù¿É£©ºÍøÇÐλµã¸½½üÉè¼ÆÒýÎïP1¡¢P2£»
b. »ØÊÕDNA£¬T4Á¬½ÓøÁ¬½Ó£¬Ê¹Ã¸ÇкóµÄDNAƬ¶Î»·»¯£»
c. »ØÊÕÁ¬½ÓºóµÄDNA£¬ÓÃÒýÎïP1¡¢P2×öPCR£¬¾Í¿ÉÀ©Ôöµ½Á½¶ËΪT-DNA²åÈëÐòÁУ¬ÖмäΪ²àÒíÐòÁÐµÄÆ¬¶Î¡£
2¡¢ÏÞÖÆÐÔÄÚÇÐøÏû»¯£ºÑ¡ÔñºÏÊʵÄÏÞÖÆÐÔÄÚÇÐø£¬»ùÒò×éÒ»¶¨ÒªÇгÉÃÖÉ¢ÐÔµÄÌõ´ø¡£ÔÚøÇÐ֮ǰ£¬Ó¦¶Ô»ùÒò×éDNA¶¨Á¿¡£
¸ù¾ÝT-DNAµÄ×ó±ß½çÐòÁÐÑ¡ÔñºÏÊʵÄøÇÐλµãEcoRI£¬BamHIºÍHindIII/PstI£¬²¢ÔÚøÇÐλµãÉÏÓθ½½üÉè¼ÆÒýÎïZ1£¬Z2£¬Z3ºÍZ4£¬È»ºóÓÃÕâЩÄÚÇÐø·Ö±ðÏû»¯»ùÒò×éDNA£¬Ã¸ÇÐÌåϵÈçÏ£º
Buffer for EcoR I         2¦Ìl
Genomic DNA         3¦Ìl
EcoR I         0.5¦Ìl £¨10u/¦Ìl£©
ddH2O         14.5 ¦Ìl
        20¦Ìl
37¶È ¹ýÒ¹£¬µçÓ¾¼ì²âøÇÐЧ¹û¡£
3¡¢»ØÊÕDNA
¢Ù ½«Ã¸ÇвúÎïתµ½1.5mlÀëÐĹÜÖУ¬ÓÃddH2OÏ´µÓ¸É¾»£¬²¢Ï¡Ê͵½250¦Ìl£»
¢Ú ¼ÓÈëµÈÌå»ýµÄ·ÓºÍÂȷ£¨V£ºV=1£º1£©£¬ÎÐÐý»ìÔÈ£¬ÊÒξ²ÖÃ1min£»
¢Û ×î´óËÙ¶È£¨13, 000 rpm£©ÀëÐÄ1min£»
¢Ü ½«ÉÏÇåÒÆµ½ÁíÒ»¹ÜÖУ¬¼ÓÈëµÈÌå»ýµÄÂȷ£¬ÎÐÐý»ìÔÈ£¬ÊÒξ²ÖÃ1min£»
¢Ý ×î´óËÙ¶È£¨13, 000 rpm£©ÀëÐÄ2min£»
¢Þ ½«ÉÏÇåÒÆµ½ÁíÒ»¹ÜÖУ¬¼ÓÈë2.5±¶Ìå»ýµÄ-20¡æÔ¤ÀäµÄÎÞË®ÒÒ´¼£¬0.1±¶Ìå»ýµÄ3M ÒÒËáÄÆ£¨pH=5.2£©£¬ÇáÇáÕñµ´»ìÔÈ£¬ÊÒξ²ÖÃ5min£»
¢ß 4¡æ×î´óËÙ¶È£¨13, 000 rpm£©ÀëÐÄ10~15min£»
¢à ÆúÉÏÇ壬¾¡Á¿³ýÈ¥¹Ü±ÚÉϵÄÒºÌ壻
¢á ¼ÓÈë1ml -20¡æÔ¤ÀäµÄ70%ÒÒ´¼£¬ÇáÇáµßµ¹¼¸´Î¡£×î´óËÙ¶È£¨13, 000 rpm£©ÀëÐÄ5min£»
¢â ³ýÈ¥ÒÒ´¼£¬ÔÚ³¬¾»Ì¨ÉÏÆ½·Å£¬½«¹Ü±ÚÉϵÄÒÒ´¼»Ó·¢µô£¬20~40¦Ìl ddH2OÈܽ⡣-20¡æ±£´æ¡£
4¡¢Á¬½Ó¡£ÌåϵÈçÏ£º
DNA        2¦Ìl
10¡Ábuffer        10¦Ìl
T4 ligase         1¦Ìl
ddH2O         87¦Ìl
        100¦Ìl
12-14¡æ   overnight
Á¬½ÓÌåϵ±È½Ï´ó£¬¶øDNAº¬Á¿±È½ÏµÍ£¬²»Ðè¼ÓÈëPEG4000£¬ÕâÑù¿ÉÒÔ´Ù½ø·Ö×ÓÄÚµÄÁ¬½Ó£¬¼õÉÙ·Ö×Ó¼äµÄÁ¬½Ó¡£ÊÔÑéÖÐÎÒÃDzÉÈ¡å¿ÎÄÓ¤µÈ£¨2002£©µÄ·½·¨£¨12-14¡æÁ¬½Ó48h£©¡£Á¬½ÓÍê³Éºó£¬65¡æ ˮԡ10minÃð»î¡£°´ÉÏÊö3.³éÌá»ØÊÕ£¬ÈܽâÓÚ40¦Ìl ddH2OÖС£
È»ºó¾Í¿ÉÒÔÓûØÊÕµÄÁ´½ÓƬ¶Î×öPCRÁË¡£
»Ø¸´´ËÂ¥

» ²ÂÄãϲ»¶

» ±¾Ö÷ÌâÏà¹ØÉ̼ÒÍÆ¼ö: (ÎÒÒ²ÒªÔÚÕâÀïÍÆ¹ã)

» ±¾Ö÷ÌâÏà¹Ø¼ÛÖµÌùÍÆ¼ö£¬¶ÔÄúͬÑùÓаïÖú:

ÒÑÔÄ   »Ø¸´´ËÂ¥   ¹Ø×¢TA ¸øTA·¢ÏûÏ¢ ËÍTAºì»¨ TAµÄ»ØÌû
лл·ÖÏí£¡
2Â¥2010-06-02 17:09:00
ÒÑÔÄ   »Ø¸´´ËÂ¥   ¹Ø×¢TA ¸øTA·¢ÏûÏ¢ ËÍTAºì»¨ TAµÄ»ØÌû

·¶¶ù

Í­³æ (СÓÐÃûÆø)

¡ï
Сľ³æ: ½ð±Ò+0.5, ¸ø¸öºì°ü£¬Ð»Ð»»ØÌû
ÕýÔÚ×ö·´Ïò£¬ÀÏÊÇ×ö²»³öÀ´£¬ÓÃÄã·½·¨ÊÔÊÔ
˵µ½²»Èç×öµ½£¬Òª×ö¾Í×ö×îºÃ£¡
3Â¥2012-11-14 21:18:21
ÒÑÔÄ   »Ø¸´´ËÂ¥   ¹Ø×¢TA ¸øTA·¢ÏûÏ¢ ËÍTAºì»¨ TAµÄ»ØÌû
Ïà¹Ø°æ¿éÌø×ª ÎÒÒª¶©ÔÄÂ¥Ö÷ shaojing690 µÄÖ÷Ìâ¸üÐÂ
×î¾ßÈËÆøÈÈÌûÍÆ¼ö [²é¿´È«²¿] ×÷Õß »Ø/¿´ ×îºó·¢±í
[Óлú½»Á÷] 3-äåßÁà¤-4-¼×È©ºÏ³É 10+3 Ñ©¸âÒ²Äܱ¬Õ¨ 2026-03-31 9/450 2026-04-06 00:24 by nBuï®
[¿¼ÑÐ] ²ÄÁÏר˶(0856) 339·ÖÇóµ÷¼Á +9 ¹þ¹þ¹þ¶ì¹þ¹þ¹þ 2026-04-05 9/450 2026-04-05 22:24 by dongzh2009
[¿¼ÑÐ] 086000ÉúÎïÓëÒ½Ò© ³õÊÔ274Çóµ÷¼Á +6 С¶£µ±À´ÁË 2026-03-30 7/350 2026-04-05 20:30 by lys0704
[¿¼ÑÐ] ÉúÎïѧ308·ÖÇóµ÷¼Á£¨Ò»Ö¾Ô¸»ª¶«Ê¦´ó£© +8 ÏàÐűػá¹ââÍòÕ 2026-04-05 10/500 2026-04-05 12:19 by Hdyxbekcb
[¿¼ÑÐ] ²ÄÁϹ¤³Ì310ר˶µ÷¼Á +13 ÀÌÀÌÎÒ¡­. 2026-04-04 14/700 2026-04-05 09:01 by À´¿´Á÷ÐÇÓê10
[¿¼ÑÐ] Çó +5 »¯¹¤×¨Ë¶323·Ö 2026-04-04 5/250 2026-04-05 08:02 by 544594351
[¿¼ÑÐ] 295Çóµ÷¼Á +4 AÄãºÃÑо¿Éú 2026-04-04 5/250 2026-04-04 22:46 by yu221
[¿¼ÑÐ] 0854Çóµ÷¼Á +4 assdll 2026-04-03 4/200 2026-04-04 22:17 by hemengdong
[¿¼ÑÐ] 294Çóµ÷¼Á +6 Grey_Ey 2026-04-02 9/450 2026-04-04 22:07 by hemengdong
[¿¼ÑÐ] µ÷¼Á +9 19945159693 2026-04-03 10/500 2026-04-04 20:16 by dongzh2009
[¿¼ÑÐ] Çóµ÷¼Á +3 ffyyu 2026-04-02 3/150 2026-04-04 19:03 by À¶ÔÆË¼Óê
[¿¼ÑÐ] Ò»Ö¾Ô¸Äϲý´óѧ324Çóµ÷¼Á +9 hanamiko 2026-03-30 9/450 2026-04-04 11:04 by Öí»á·É
[¿¼ÑÐ] ²ÄÁϵ÷¼Á +11 ÎâèùÓ±£¡ 2026-04-03 11/550 2026-04-04 09:56 by ССÊ÷2024
[¿¼ÑÐ] 0856µ÷¼Á +8 ÇúÌýóÞ 2026-03-30 8/400 2026-04-04 08:46 by tianyyysss
[¿¼ÑÐ] 282Çóµ÷¼Á +20 ycy1201 2026-04-01 22/1100 2026-04-04 00:42 by userper
[¿¼ÑÐ] ³õÊԳɼ¨337ÕÒµ÷¼Á +3 ??? ?. ? 2026-04-03 3/150 2026-04-03 11:43 by ÍÁľ˶ʿÕÐÉú
[¿¼ÑÐ] ±§Ç¸ +5 ÌïºéÓÐ 2026-03-30 5/250 2026-04-03 10:24 by linyelide
[¿¼ÑÐ] ÉúÎïѧ308·ÖÇóµ÷¼Á£¨Ò»Ö¾Ô¸»ª¶«Ê¦´ó£© +6 ÏàÐűػá¹ââÍòÕ 2026-03-31 7/350 2026-04-02 23:16 by JourneyLucky
[¿¼ÑÐ] Ò»Ö¾Ô¸±±½»´ó²ÄÁϹ¤³Ì×Ü·Ö358 +3 cs0106 2026-04-02 5/250 2026-04-02 11:37 by olim
[¿¼ÑÐ] 354Çóµ÷¼Á +4 lxb598 2026-03-31 5/250 2026-04-02 09:55 by Jaylen.
ÐÅÏ¢Ìáʾ
ÇëÌî´¦ÀíÒâ¼û