| | |||||||
| Register | Search | Today's Posts | Mark Forums Read |
| Molecular Biology Techniques Molecular Biology Forum. Includes forums for common molecular biology techniques. |
| | LinkBack | Thread Tools | Display Modes |
|
#1
| |||||||||||
| |||||||||||
| Good day. i failed to clone a 2.2 kb gene in to a vector (about 12 kb). anybody can give me some oppinion and suggestion? below are the details vector: TAKARA pRI201-AN. cut with SacI, treated with T4 DNA polimerase to produce blunt ends, the cut with NdeI to product a linear vector with sticky end at one side and blunt end at another side. gel purified insert: PCR product amplified with Pfu dna polymerase ( blunt end), cut with NdeI at to produce stiky end at one side. ge purified ligation: 90ng of vector and about 47ng of insert. (vector: insert =1:3) in 20ul reaction Transformation: heat shock Result: all of the colonies i obtained are with out insert, seem like the vector ligate with another vector ( sticky end bind with sticky end, blunt end with blunt) I really hpe somebody can help me to solve this problem. Thank you very much. |
| Tags |
| cloning , fragment , large |
| Thread Tools | |
| Display Modes | |
|
|
| | ||||
| Thread | Thread Starter | Forum | Replies | Last Post |
| Vector for large insert cloning | Putra Taufik | Molecular Cloning Forum | 5 | 11-18-2010 08:06 AM |
| Human Cytome Project - Update 24 Jan. 2005 | Peter Van Osta | Cell Biology and Cell Culture | 1 | 08-01-2010 02:18 PM |
| Problems Cloning Large DNA Fragments Together | tonyz06 | Molecular Cloning Forum | 0 | 02-07-2008 09:48 AM |
| Human Cytome Project - Update 6 Jan. 2005 | Peter Van Osta | Cell Biology and Cell Culture | 0 | 01-06-2005 10:18 AM |
| GATEWAY cloning of large PCR fragments | Ricky Boernke | Protocols and Methods Forum | 1 | 09-05-2004 11:50 PM |