Are there protocols out there, that could help me out with that task? Expression should be in human cells under the control of a minimal promoter, assay for promoter activity would ideally be GFP.
I suppose with whole genome cloning you mean would like to build a cDNA library? There are several protocols out there, but generally commercial cloning systems like the Gateway system from Life technologies (formerly Invitrogen) do a good job.
Here is a link to their site, I hope that is helpful:
http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cloning/cDNA-Libraries-and-Library-Construction.html
Great ... thanks ... that's already a good hint ...
Though ... actually I'm aiming for the not so easy things ... no cDNA ... I'm thinking whether it's possible to clone the whole genome into a library ... introns, exons and all the intergenic regions too ... just for instance sonicate the genome into peaces like you do it with ChIP ... and than clone everything into a plasmid library ... I know ... pretty ambitious ... and I'm curious of your comments ...
It is good, clear concise answer. More of an interest in Pharmacology and kinetics/ dynamics in personal opinion, but all the same it should link with a question in the research foraym. McNabb
http://www.researchgate.net/publictopics.PublicTopicRewriteHandler.html?params=%2FGenetic_Engineering%2F
This is called "shotgun cloning" or "construction of shotgun libraries"--and you'll find many Google hits with those terms. This is, for instance, how the libraries of DNA fragments used to perform the initial sequencing of the human genome were prepared. As with most things, the method of choice depends on the intended purpose. Do you want very large segments containing full length genes or do you want small fragments which might have detectable promoter activity if cloned into an expression vector?
Hi David ... thanks for your reply ... actually I would like to perform an enhancer screening with little fragments ... so in the end it's all about choosing the right plasmid for that purpose ... one that expresses a GFP reporter for instance having a minimal promoter in front ... any suggestions ... ?
THanks for your help ...!
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning