Single colony purification by streaking - (Dec/18/2008 )
I want to check the expression of my clone but i have been asked to colony purify by streaking for single colony.
So fiest i will transofrm into BL21 (DE3) cells and after getting the colony take some of them and grow overnigh culture and streak again on the LB media to get single colony and that single colony can be used for expression. Is it like this?????
Even after the transformation, you will get single colony as there will be only few transformants. So, probably to perform the transformation, you should pick the single colony.
I think you should go back and ask the person exactly what they meant. Normally if your clone is already in cells, then I would just streak it onto a plate and pick from there rather than go through the whole transformation procedure again. But if you do decide to do the retransformation, then you can probably skip the overnight liquid culture in between streaking.
Yes, agreed. Normally after transformation I select a colony and restreak directly from the colony (no liquid culture). To make this easier, you can resuspend it in a small amount of media. Then I repeat this for a few generations (3-4 times).
Yes, agreed. Normally after transformation I select a colony and restreak directly from the colony (no liquid culture). To make this easier, you can resuspend it in a small amount of media. Then I repeat this for a few generations (3-4 times).
Your answer is very good, this is relevant to me too but i could not understand what do you mean by\'Then I repeat this for a few generations (3-4 times)."