1402 Quick Cloning Kit
User Manual For Quick Cloning Kit
²úÆ·Ãû³Æ |
Cat. No. |
¹æ¸ñ |
×é·Ö |
1402 Quick Cloning Kit |
1402-1 |
50 rxns |
1 tube of 4X Cloning Enzyme Premix |
1402-5 |
250 rxns |
5 tubes of 4X Cloning Enzyme Premix |
´¢´æÓÚ -20?C
¸ÅÊö
±¾²úÆ·ÀûÓÃÌØÊâµÄÖØ×éÔÀí£¬²»ÒÀÀµÓÚÁ¬½Óø£¬¿ÉʵÏÖ¼òµ¥¡¢¿ìËÙ¡¢¸ßЧµÄDNA¶¨Ïò¿Ë¡¡£ÔØÌå¿Éͨ¹ýÈÎÒâ·½·¨ÏßÐÔ»¯£¬È»ºóÓëÁ½¶Ëº¬ÓÐ15~25bpÔØÌåÍ¬Ô´ÇøÓòµÄPCRƬ¶ÎÖØ×飬·´Ó¦Ê±¼ä¶Ì£¬ÖØ×éЧÂÊ>95%£¬²¢¿ÉʵÏÖ¶àÖÁ5¸öƬ¶ÎµÄ¸ßЧÎÞ·ì¿Ë¡¡£
ÓÅÊÆ
♦ ¿É²åÈëÈÎÒâÔØÌåµÄÈÎÒâλÖÃ
♦ Ò»²½£¬¼òµ¥£¬×¼È·£¬¿ìËٵؽøÐÐ¶àÆ¬¶Î¶¨Ïò¿Ë¡£¬·´Ó¦½öÐè15min£¬Ð§Âʸߴï95%
♦ ²»ÒÀÀµÓÚÁ¬½Óø£¬PCR²úÎïÎÞÐèøÇУ¬ÎÞÐèÁ×Ëữ
♦ ÎÞ·ìÁ¬½Ó£¬²»ÒýÈë¶îÍâÐòÁÐ
¿Ë¡ʵÑéÁ÷³Ì
I: ÔØÌåÏßÐÔ»¯£¬¿ÉøÇлòͨ¹ýPCR·½·¨
II: Ä¿µÄƬ¶ÎÖÆ±¸£¬PCR²úÎï5’ºÍ 3’×îÄ©¶ËµÄÐòÁзֱðºÍÏßÐÔ»¯¿ËÂ¡ÔØÌåÁ½Ä©¶ËÐòÁÐÍêȫһÖÂ
III: ÔØÌ壬ĿµÄƬ¶Î°´Ä¦¶û±È~1:3Óë¿Ë¡ÊÔ¼Á»ìÔÈ£¬50°C·õÓý15~30min
IV: ·´Ó¦²úÎïת»¯
¾ßÌå¿Ë¡ʵÑé²Ù×÷
I: ÔØÌåÏßÐÔ»¯
ÏßÐÔ»¯ÔØÌåÊǿˡ³É¹¦µÄÖØÒªÇ°Ìᣬ¿Éͨ¹ýøÇлñµÃ£¬Æ½Ä©¶Ë»òÕßճĩ¶Ë¾ù¿É£¬Ë«Ã¸ÇиüÓÐÀûÓÚ½µµÍ±³¾°;Ò²¿ÉÌṩPCRÀ©Ôö»ñµÃ£¬¸ß±£ÕæDNA¾ÛºÏø¿É±ÜÃâ²úÉú²»±ØÒªµÄÍ»±ä¡£
II: ÒýÎïÉè¼Æ
Ä¿µÄƬ¶ÎÀ©ÔöËùÐèµÄÕý·´ÏòÒýÎï³ýº¬ÓÐÓë×ÔÉíÆ¥ÅäµÄÌØÒìÐÔÐòÁÐÍ⣬¾ùÐ躬ÓÐÓëÔØÌåÄ©¶ËͬԴµÄ15~25bpÖØµþÐòÁУ¬ÈçÐèÌí¼ÓøÇÐλµã£¬½ÓÍ·ÐòÁУ¬±êÇ©ÐòÁеȣ¬¿ÉÌí¼ÓÔÚ¶þÕßÖ®¼ä¡£¼´£º
PCR Forward Primer = ÔØÌåÕýÏòoverlapÐòÁÐ(15~25bp)+ÖмäøÇÐλµãµÈÐòÁÐ+Ä¿µÄƬ¶ÎÕýÏòÌØÒìÐÔÐòÁÐ(20~25bp)
PCR Reverse Primer = ÔØÌå·´ÏòoverlapÐòÁÐ(15~25bp)+ÖмäøÇÐλµãµÈÐòÁÐ+Ä¿µÄƬ¶Î·´ÏòÌØÒìÐÔÐòÁÐ(20~25bp)
ÈôÒª¿Ë¡¶à¸öÄ¿µÄƬ¶Î£¬Æ¬¶Î¼äÒýÎïÉè¼ÆÔÀíÓëÉÏÏàͬ¡£
III: Ä¿µÄƬ¶ÎÖÆ±¸
Ä¿µÄ²úÎïÐèÑ¡Óø߱£ÕæDNA¾ÛºÏø½øÐÐÀ©Ôö£¬ÓÉÓÚPCRÒýÎïͨ³£½Ï³¤£¬ÍË»ðζȿɸù¾ÝÌØÒìÐÔÇøTmÖµ¼°¾ÛºÏø±¾ÉíÈ·¶¨¡£
IV: ¿Ë¡·´Ó¦Ìåϵ
Recommended Amount |
|
4X Cloning Enzyme Premix |
5 μl |
Linearized vector |
40~80ng |
Inserts |
ÔØÌångÊý*3*Ƭ¶Î´óС(bp)/ÔØÌå´óС(bp)=ng |
Total Volume |
20 μl |
·´Ó¦ÎïÇáÇá»ìÔȺó£¬50°C 15~30min¡£·´Ó¦½áÊøºó¿ÉÖ±½ÓÓÃÓÚºóÐø×ª»¯»òÕß-20°C±£´æ±¸Óá£
×¢£º
1)ÖØ×鯬¶ÎΪ2~3¶Îʱ£¬ÔØÌåÓë¸÷Ƭ¶Î¼ÓÈëÁ¿Ô¼Îª0.02 – 0.5 pmols£¬ÖØ×鯬¶ÎΪ4~6¶Îʱ£¬ÔØÌåÓë¸÷Ƭ¶Î¼ÓÈëÁ¿Ô¼Îª0.2 – 1 pmols£¬ÔØÌåÓë¼ÓÈëÄ¿µÄƬ¶ÎµÄ×î¼ÑĦ¶û±ÈΪ1:3
2)ÖØ×鯬¶Î½Ï¶àʱ£¬¿ÉÊʵ±ÑÓ³¤·´Ó¦Ê±¼äÖÁ1h

V: ת»¯
1. °Ñ¸ÐÊÜ̬ϸ°ûÖÃÓÚ±ùÖнⶳ;
2. ¼ÓÈëÓÃÓÚת»¯µÄÖØ×é·´Ó¦²úÎï20μl £¬±ùÖзÅÖÃ30minºó£¬42¡æ Èȼ¤60¡«90s;
3. ¼ÌÐø±ùÉÏ·ÅÖÃ2min;
4. Ìí¼Ó 500 μl S.O.C »òÕß LBÅàÑø»ù£¬ 37?C Ò¡´²ÅàÑø 1 h;
5. ½«×ª»¯¾úÒºÀëÐĺóÆúÉÏÇ壬ÇáÇὫ ³ÁµíÐü¸¡ºó¾ùÔÈÍ¿²¼ÓÚº¬ÓÐÊÊÒË¿¹ÐÔµÄLBƽ°åÉÏ;
6. 37?C µ¹ÖùýÒ¹ÅàÑø
×¢ÒâÊÂÏî
Problem |
Probable cause |
Solution |
ת»¯ºó¾úÂäºÜÉÙ»òÕßûÓÐ |
DNAŨ¶ÈÌ«µÍ |
ʹÓÃÍÆ¼öµÄDNAÓÃÁ¿. |
Ä¿µÄƬ¶Î»òÔØÌåÖк¬ÓÐÎÛȾ. |
PCR²úÎï¼°ÏßÐÔ»¯ÔØÌå¾ùÐè´¿»¯ |
|
¸ÐÊÜ̬ϸ°ûЧÂÊÌ«µÍ |
ʹÓÃЧ¼Û>1×108cfu/µg µÄ¸ÐÊÜ̬ϸ°ûЧ¹û¸üºÃ |
|
ÔØÌåÏßÐÔ»¯²»ÍêÈ« |
ÔØÌåÇнº»ØÊÕ |
|
ÑôÐÔ¿Ë¡½ÏÉÙ£¬¶àΪ¿ÕÔØÌå |
±»º¬ÓÐÏàͬ¿¹ÐÔµÄÔØÌåÎÛȾ |
PCR²úÎïÖпÉÄܺ¬ÓÐÄ£°åÖÊÁ££¬ÍƼöÇнº»ØÊÕ |
ƽ°å¿¹ÐÔ´íÎó |
È·±£Æ½°åÐÂÏÊÅäÖÆÇÒÌí¼ÓÕýÈ·¿¹ÉúËØ. |
|
ÔØÌåøÇв»ÍêÈ« |
øÇÐʱ¼äÑÓ³¤¿ÉÔö¼ÓÏßÐÔ»¯³Ì¶È£¬½µµÍ±³¾° |
ÉÏÒ»Ò³
ÏÂÒ»Ò³
ÉÏÒ»Ò³
ÏÂÒ»Ò³
ÁôÑÔ×Éѯ
ÐèÒªÁË½â¹ØÓÚjxf¼ªÏé·»¹ÙÍøÈ«ëĵĸü¶àÐÅÏ¢ºÍ²úÆ·£¬Äú¿ÉÒÔͨ¹ýÁôÑÔ»òµç»°Óëjxf¼ªÏé·»¹Ù·½appÈ¡µÃÁªÏµ£¬jxf¼ªÏé·»¹Ù·½app»áΪÄúÌṩרҵµÄ·þÎñÓë²úÆ·¡£»¶Ó²¦´ò·þÎñÈÈÏߣº17373366007£¨Êг¡²¿»Æ¾Àí£©