| |||||||
| Register | Search | Today's Posts | Mark Forums Read |
| Molecular Cloning Forum DNA cloning forum. Discuss the cloning of plasmids, vectors, DNA, RNA, cDNA, proteins, and other molecules for expression, sub-cloning, and other methods. Also discuss digestion, ligation, transformation, plasmid preparation, cDNA library screening, and competent cells here. |
 |
| | LinkBack | Thread Tools | Display Modes |
|
#1
02-22-2011, 04:26 AM
| |||||||||||
| |||||||||||
 Is it the transformation or the plasmid? I've been trying to transform E.coli cells (JM109, XL-1 BLue and DH5a) with a pET15b vector. The results have been sketchy - only got a single colony of XL-1 Blues on ampicillin plates. The cells grew fine in liquid medium with amp and a miniprep yielded on okay amount of plasmid. However, the company I've sent it to for sequencing cannot find anything: their pcr isn't picking up anything and nothing is seen on a gel. WTH? Is it possible that the transformation was unsuccessful - and if so, what came out in the miniprep? Or, the plasmid has been slightly modified but has been successfully used by another group, so I think its unlikely to be that. Finally - do you think the problem could be with the sequencing??  |
|
#2
02-22-2011, 11:40 AM
| |||||||||||
| |||||||||||
 Re: Is it the transformation or the plasmid? When you say you got a decent amount of plasmid but nothing on a gel, how did you test the success of your miniprep? |
|
#3
06-18-2012, 06:47 AM
| |||||||||||
| |||||||||||
| Re: Is it the transformation or the plasmid? I also want to know |
|
#4
03-08-2013, 07:13 PM
| |||||||||||
| |||||||||||
| Re: Is it the transformation or the plasmid? So no one knows the answer to this? I have been trying to clone into a pet15b vector as well (Nde1 and BamH1 sites) and it just won't go in. Does anyone know what's going on? |
|
| Tags |
| pet15b , plasmid , transformation |
| Thread Tools | |
| Display Modes | |
|
|
Similar Threads | ||||
| Thread | Thread Starter | Forum | Replies | Last Post |
| pENTR1A Transformation not working | CambridgeBiochemist | Molecular Cloning Forum | 1 | 06-18-2012 06:53 AM |
| plasmid transformation using yeast | davidfranco72 | Yeast Forum | 9 | 06-18-2012 06:17 AM |
| Transformation of PCR product to get resistance with no plasmid | leviuso | Yeast Forum | 1 | 07-14-2009 12:40 PM |
| Transformation of Bacteria by Plasmid DNA | domba | Molecular Biology Articles and Protocols | 1 | 08-12-2008 01:37 PM |
| What is the different between the plasmid that used to overexpressprotein in E coli and that for Agrobacterium to transformation plants? | guo shukui | Arabidopsis and Plant Biology | 0 | 12-01-2006 04:33 PM |
Linear Mode
