|
¡ï ¡ï ¡ï ¡ï ¡ï dhd997(½ð±Ò+2):¸Ðл»ØÌû½»Á÷ 2010-11-21 10:09:25 lstt09nk(½ð±Ò+3):ÔÚ×Ô¼ºµÄÌû×ÓÀï°ïÖú±ðÈË~ 2010-11-26 09:37:00
ÎҵĹý³ÌÏà¶Ô¼òµ¥£¬ÓÃcDNA£¬Ê¡ÁËøÇеIJ¿·Ö£¬Èç¹ûÊÇÓÃDNAµÄ»°£¬ÎÒÕâÓиö×ÊÁÏ£¬Ò²ÊÇСľ³æ¿´µ½µÄ£º·´ÏòPCR £¨inverse-PCR£©ÊµÑé²½Öè
inverse-PCRÊÇ¿Ë¡²åÈëƬ¶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·´ÏòPCRÊÇ¿Ë¡T-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¬ÖмäΪ²àÒíÐòÁеÄƬ¶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´óËٶȣ¨13, 000 rpm£©ÀëÐÄ1min£»
¢Ü ½«ÉÏÇåÒƵ½ÁíÒ»¹ÜÖУ¬¼ÓÈëµÈÌå»ýµÄÂȷ£¬ÎÐÐý»ìÔÈ£¬ÊÒξ²ÖÃ1min£»
¢Ý ×î´óËٶȣ¨13, 000 rpm£©ÀëÐÄ2min£»
¢Þ ½«ÉÏÇåÒƵ½ÁíÒ»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´óËٶȣ¨13, 000 rpm£©ÀëÐÄ10~15min£»
¢à ÆúÉÏÇ壬¾¡Á¿³ýÈ¥¹Ü±ÚÉϵÄÒºÌ壻
¢á ¼ÓÈë1ml -20¡æÔ¤ÀäµÄ70%ÒÒ´¼£¬ÇáÇáµßµ¹¼¸´Î¡£×î´óËٶȣ¨13, 000 rpm£©ÀëÐÄ5min£»
¢â ³ýÈ¥ÒÒ´¼£¬ÔÚ³¬¾»Ì¨ÉÏƽ·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¡£
±¾ÎÄÀ´×Ô: Сľ³æÂÛ̳ http://muchong.com/bbs/viewthread.php?tid=2109736 |
|