Hello
we are trying to do some GATEWAY cloning backwards. We want to recover a destination clone from an expression clone - simple, I would have said 4 weeks ago. We take the expression clone (amp), add pDONR207 and make a BP reaction. After transforming into DB3.1 we select for amp and chloramphenical.
We get only colonies that carry both original plasmids. Sounds logical - assuming that both plasmids can be replicated in a single cell (I think i had heard previously that this is not neccessarily the case) and that transformation efficiency is high (quite possible).
We tried digesting the pDONR207 backbone before BP (I admit the digest was not perfectly complete). The results got no better at all.
Are we completely on the wrong track - or what is going on? Is our strategy in principle OK - do we just have to screen harder? Many
thanks for you advice, Henrik
Cloning is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects, plants or animals reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments (molecular cloning), cells (cell cloning), or organisms (organism cloning). Beyond biology, the term also refers to the production of multiple copies of a product such as digital media or software.
regards
Dear Khalid,
I am not sure how this relates to our Gateway problem. Could you explain what you mean exactly?
Henrik
- Badges
- Science method