²é¿´: 2461  |  »Ø¸´: 0

swknptb429

гæ (³õÈëÎÄ̳)

[½»Á÷] ¶¨µãÍ»±äµÄ³£¼ûÎÊÌâ¼°²ßÂÔ-BioEngX·¢²¼

¶¨µãÍ»±ä£¨site-directed mutagenesis£©²¢²»ÊÇÒ»¸ö¸´ÔÓµÄʵÑ飬Ȼ¶øÓÐʱºòÎÒÃÇÔÚʵÑéÖÐÈ´»áÓöµ½¸÷ÖÖ¸÷ÑùµÄÎÊÌ⡣һЩ³£¼ûµÄÎÊÌâ°üÀ¨µ«²»ÏÞÓÚ: ¾úÂäÌ«¶à£»Ã»ÓоúÂäÉú³¤£»µÃµ½¾úÂ䣬µ«¾úÂäÖв»º¬ÓÐÄ¿µÄÍ»±äµãµÈ¡£

ÈçºÎ·ÖÎö³öÏÖÎÊÌâµÄÔ­Òò²¢½â¾öÕâЩÎÊÌâÄØ£¿
С±àÄÜÌṩµÄ×îºÃµÄ½¨Òé¾ÍÊÇÔÚ½øÐж¨µãÍ»±äʵÑéʱһ¶¨ÒªÓжÔÕÕ×飨control£©¡£Ðí¶à¶¨µãÍ»±äÊÔ¼ÁºÐÖж¼»á°üº¬¶ÔÕÕÓõÄÄ£°å£¬ÒýÎïÒÔ¼°×ª»¯Ê±ºòµÄ±ê×¼ÖÊÁ£¡£¶ÔÕÕʵÑéÄܹ»°ïÖúÄã¸üÈÝÒ×µØÈ·¶¨¿ÉÄܳöÏÖÎÊÌâµÄʵÑé²Ù×÷²½Ö裬½ÚÊ¡ÄãµÄʱ¼äÒÔ¼°ÊÔ¼ÁºÄ²Ä¡£
ÏÂÃæÊÇһЩÔÚ¶¨µãÍ»±äʵÑé¹ý³ÌÖнâ¾ö³£ÓöÎÊÌâµÄС¼¼ÇÉ£º
µ±Æ½°åÉϾúÂä(colonies)Ì«¶àʱ:
1¡¢½µµÍPCR·´Ó¦ÖеÄÄ£°å£¨template£©Å¨¶È
2¡¢¼õÉÙת»¯Ê±PCR²úÎïµÄÓÃÁ¿
½«²»Í¬Ìå»ýµÄת»¯ºó²úÎïÍ¿ÔÚÆ½°åÉÏ£¬±ÈÈç10 ?l, 20 ?l, 50 ?l, 100 ?l¡£Ñ¡È¡¾úÂä·Ö²¼×îºÏÊÊ£¨well-spaced£©µÄƽ°åÌôÑ¡ÑôÐÔ¿Ë¡¡£  
3¡¢ÑÓ³¤Dpn IøÇУ¨digestion£©Ê±¼ä£¬±ÈÈçøÇÐ2¸öСʱ£¬´úÌæ1¸öСʱ

µ±Æ½°åÉÏûÓоúÂäʱ:
1¡¢Ìá¸ßPCR·´Ó¦ÖÐÄ£°åµÄÓÃÁ¿
2¡¢Ìá¸ßת»¯Ê±PCR ²úÎïµÄÓÃÁ¿
3¡¢Èç¹ûDNAµçÓ¾ÖÐÏÔʾPCR ²úÎïŨ¶È²»¸ß»òPCRʧ°Ü£¬¿ÉÒÔÊÔÓò»Í¬µÄζÈÌݶȽøÐÐPCR ·´Ó¦
4¡¢¸Ä±äPCR·´Ó¦µÄÑÓÉìʱ¼ä¼°Î¶ȣ¨±ÈÈç½µµÍζȵ½68 oC, ÒÔ60 s /KbµÄ±ÈÀýÉèÖÃÑÓÉìʱ¼ä£©
5¡¢PCR·´Ó¦Ê±¼ÓÈëÒ»µã DMSO (2-8%)£¬ÓÃÀ´¸¨Öú¸»º¬GCµÄÇøÓòË«Á´µÄ·ÖÀë
6¡¢¼ì²é¸ÐÊÜ̬£¨competent cells£©Ï¸°ûÊÇ·ñÓÐЧ£¨ÀûÓöÔÕÕÖÊÁ££©
7¡¢Å¨ËõøÇкóµÄDNA£¬¿ÉÒÔÀûÓÃÒÒ´¼£¨Ethanol£©³Áµí£¨precipitate£©Ã¸ÇйýµÄDNA, È»ºóÔÚת»¯Ç°ÓøüСÌå»ýµÄÒºÌåÖØÐü£¨re-suspend£©Ö®
8¡¢×ª»¯Ç°´¿»¯DNAÑùÆ·£¬È¡³öPCR·´Ó¦ÖеÄÑÎÀë×Ó
¾úÂäÖÐûÓÐÄ¿µÄÍ»±äÐÍ(desired mutation):
1¡¢ÀûÓú¬ÓÐDam¼×»ù»¯Ã¸£¨methylase£©µÄ¾úÖêÀ©ÔöÄ£°åÖÊÁ££¬±ÈÈçJM109£¬DH5¨»¡£¿ÉÄÜÓÐЩͬѧ²»Ã÷°×ÕâΪºÎÒ⣬´ýС±àÒ»Ò»µÀÀ´¡£Dam¼×»ù»¯Ã¸¿É½«GATCÐòÁÐÖеÄÏÙàÑßÊN6λµã½øÐм׻ù»¯ÐÞÊΣ¬¶øÕâһλµãǡǡÊÇDpnI øµÄʶ±ðλµã¡£Òò´Ë²»ÄÜÓ¦ÓÃDam¼×»ù»¯Ã¸È±Ê§µÄ¾úÖêÉú²úÄ£°åÖÊÁ££¬±ÈÈçJM110 ¾úÖê¡£ÕâÖÖ¾úÖê²úÉúµÄDNAÎÞ·¨±»DpnIøÇеô£¬×ª»¯DNAÖк¬ÓÐÒ°ÉúÐÍÄ£°å£¬½«²úÉú´óÁ¿µÄ¼ÙÑôÐÔ£¨fake positives£©¡£
Ìá¸ßDpnI øÇÐʱ¼ä»òÕßÌá¸ßDpnIøµÄÓÃÁ¿
2¡¢¼õÉÙPCRÑ­»·Êý£¬¹ý¶àµÄPCRÑ­»·¿ÉÄܵ¼Ö·ÇÌØÒìÐÔÍ»±äµãµÄÒýÈë
3¡¢Èç¹ûÉÏÊö²ßÂÔÈÔ²»Äܽâ¾öÎÊÌ⣬ÄÇÖ»ÄÜÖØÐÂÉè¼ÆÒýÎïÁË£¬ÓйØÒýÎïÉè¼ÆÎÊÌ⣬Ïê¼ûÀúÊ·ÐÅÏ¢

·¢×ÔСľ³æIOS¿Í»§¶Ë
»Ø¸´´ËÂ¥
ÒÑÔÄ   »Ø¸´´ËÂ¥   ¹Ø×¢TA ¸øTA·¢ÏûÏ¢ ËÍTAºì»¨ TAµÄ»ØÌû
Ïà¹Ø°æ¿éÌø×ª ÎÒÒª¶©ÔÄÂ¥Ö÷ swknptb429 µÄÖ÷Ìâ¸üÐÂ
×î¾ßÈËÆøÈÈÌûÍÆ¼ö [²é¿´È«²¿] ×÷Õß »Ø/¿´ ×îºó·¢±í
[»ù½ðÉêÇë] ¹ú×ÔÈ»ÃæÉϸ´ÅÌ~»¶Ó­ÌÖÂÛ +5 ÇçÌì¼ÓÓÍ 2026-08-26 6/300 2026-08-27 04:49 by littlezl
[½Ìʦ֮¼Ò] ÊÛSCIÒ»ÇøÎÄÕ£¬ÎÒ:8.O.551.O.5.4,¿ÆÄ¿È«,¿ÉÙ¤¼± +3 LIbGuocjEEYw 2026-08-26 4/200 2026-08-27 04:33 by Ie9AyIAvGbvs
[»ù½ðÉêÇë] ¿´°åÉÏÕâô¶àÖеģ¬ÓеãÏñ50ÈËȺÀï49¸öÈ˶¼ÊÇÆ­×ÓµÄÄÇÖָоõ¡­¡­ +3 a089 2026-08-26 3/150 2026-08-26 23:21 by young90
[»ù½ðÉêÇë] 2026ÄêµÄ¹ú¼ÒÉç¿Æ»ù½ðÏîĿͨѶÆÀÉóµÄйæÔòÓëж¯Ïò¡¢ÐÂÌôÕ½ +7 process2012 2026-08-23 10/500 2026-08-26 19:23 by hmhminy
[»ù½ðÉêÇë] Ôõô²é°¡ +5 huang1991js 2026-08-26 5/250 2026-08-26 16:09 by Equinoxhua
[»ù½ðÉêÇë] ³öÀ´ÁË +9 trojank 2026-08-26 9/450 2026-08-26 14:25 by ±¦±´³æ×Ó
[»ù½ðÉêÇë] Ϊʲô¹ú×ÔÈ»²»ÄÜÖ±½Ó¹«²¼ +4 bjdxyxy 2026-08-26 4/200 2026-08-26 13:12 by qingmu1201
[»ù½ðÉêÇë] ¹úºÏÀïÃæÄÜ¿´µ½ÁË +7 Ò»»³Ü°Çï 2026-08-26 7/350 2026-08-26 11:23 by zhaosm1982
[»ù½ðÉêÇë] ÏîÄ¿ÐÅÏ¢ºÍ¾­·ÑÐÅÏ¢ÔÚϵͳÀï¶¼¿ÉÒÔ¿´µ½ÁË +6 wittyboy 2026-08-26 14/700 2026-08-26 10:55 by wittyboy
[»ù½ðÉêÇë] ¹úºÏ¿É²éÁË +3 paperzjh 2026-08-26 3/150 2026-08-26 10:41 by LemmonTr
[Ö°³¡ÈËÉú] ѧÉúײ¼û¸¨µ¼Ô±ËÍÍâÂô£¬µÚ¶þÌìÈ«°à¶¼³ÁĬÁË +3 ¾¨ÓãÈÚ½ð_Õã½­_É 2026-08-22 3/150 2026-08-26 09:01 by zzuzxg
[»ù½ðÉêÇë] Å£À´£¡Ã×À´£¡ÃæÀ´£¡ +8 beefly 2026-08-26 8/400 2026-08-26 08:37 by xuzhipiao
[»ù½ðÉêÇë] ÔÚ¼á±ù»¹¸Ç×ű±º£µÄʱºò£¬ÎÒ¿´µ½ÁËÅ­·ÅµÄ÷»¨¡£ (½ð±Ò+10) +6 ziyangfang 2026-08-25 9/450 2026-08-25 20:26 by huagongfeihu
[»ù½ðÉêÇë] ½ñÈÕ²»·Å°ñ£¿Íø´«¹ú×ÔȻԤ¼Æ 8 Ô 27 Èտɲé½á¹û +17 ҽѧÀÏÄк¢ 2026-08-20 22/1100 2026-08-25 15:36 by ҽѧÀÏÄк¢
[»ù½ðÉêÇë] Èç¹û´Ë¿ÌÄãÕýÔÚΪ¹ú»ù¸Ðµ½½¹ÂÇ£¬²»·ÁÀ´ÌýÌýÕâÊס¶»ù½ðÖ®Íâ¡· +8 scalable 2026-08-24 8/400 2026-08-25 12:52 by jnhyjjm
[»ù½ðÉêÇë] ûÓÐÈκÎÏûÏ¢-ÊDz»ÊǾÍÁ¹ÁË +9 ͼÀ²Í¼À² 2026-08-24 10/500 2026-08-25 11:59 by ÄϺ£Ð¡¸ç
[»ù½ðÉêÇë] ÎÒÃæÉÏÍêµ°ÁË +13 ÇÒÌý»¢Ð¥ 2026-08-20 14/700 2026-08-25 09:10 by mrkang
[»ù½ðÉêÇë] ÈÃÎÒÖÐÒ»¸öÃæÉϰɣ¡ +13 ´óƼ1987 2026-08-20 16/800 2026-08-24 10:23 by ̫ɵÁË
[»ù½ðÉêÇë] ʱ¼ä´Á½ñÌ죬20ºÅ±äÁË +5 archvillain 2026-08-20 5/250 2026-08-22 06:12 by hui_daxiao
[»ù½ðÉêÇë] ʱ¼ä´ÁÓÖ±äÁË +13 wuchongjun 2026-08-20 19/950 2026-08-21 17:21 by ×Ïɼ´¼
ÐÅÏ¢Ìáʾ
ÇëÌî´¦ÀíÒâ¼û