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1402 Quick Cloning Kit


User Manual For Quick Cloning Kit

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Cat. No.

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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

 

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♦ ¿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

 

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Problem

Probable cause

Solution

ת»¯ºó¾úÂäºÜÉÙ»òÕßûÓÐ

DNAŨ¶ÈÌ«µÍ

ʹÓÃÍÆ¼öµÄDNAÓÃÁ¿.

Ä¿µÄƬ¶Î»òÔØÌåÖк¬ÓÐÎÛȾ.

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