why must we use freshly streak but not old/spread bacteria? - (Jan/11/2011 )
To prepare competent cells,
What is the reason of using streak colony than spread colony?
Why must the colony be fresh?
Why can't i directly put old bacteria colony or Ecoli powder in LB for O/N culture instead of streaking followed by O/N culture?
First: to get a single colony to start from.
Second: they will grow faster.
By the way, are you sure to use ON culture? Because we usually grow them till they reach an OD of 0.8 which takes about 3-4 hours.
ElHo on Tue Jan 11 16:29:16 2011 said:
First: to get a single colony to start from.
Second: they will grow faster.
By the way, are you sure to use ON culture? Because we usually grow them till they reach an OD of 0.8 which takes about 3-4 hours.
I am using Fermentas transform aid kit, which protocol is O/N culture.
I thought spreading will produce single colony as well?
The most important thing is to pick a single colony to start with. You are right, spreading also creates single colonies but you usually have to plate several dilutions of your bacterial suspension on different plates to end up with single colonies that are easy to pick. By streaking out bacteria on one plate itīs far more easy to end up with single colonies. I checked the protocol of your kit because I was very surprised about using ON culture for preparing competent cells. The next step after ON culture is to inocculate a new culture for 20 min. So that makes more sense!
ElHo on Thu Jan 13 12:13:43 2011 said:
The most important thing is to pick a single colony to start with. You are right, spreading also creates single colonies but you usually have to plate several dilutions of your bacterial suspension on different plates to end up with single colonies that are easy to pick. By streaking out bacteria on one plate itīs far more easy to end up with single colonies. I checked the protocol of your kit because I was very surprised about using ON culture for preparing competent cells. The next step after ON culture is to inocculate a new culture for 20 min. So that makes more sense!
Sorry that i've forgot to mention inoculation for another 20 minutes. Why is it important to use single colony instead of more than 1 colony? I have very poor streaking skill and hence i spread. I didn't do the dilution part but it is possible to pick single colony from my plate. And my spread plate's colony is easier to pick than streak plate
IS there any book that i can read regarding the principles of steps in cloning, such as those that i've ask?
By starting from a single colony one can be sure that each cell in your culture is descendend from a single founder cell, so each of your cells will have the same genetic background. If you get single colonies by plating, that's ok. I personally find it more convenient to produce single colonies by streaking, especially when not working with liquid cultures. Streaking is not difficult, you might try it. Current protocols in molecular biology was a great help to me when I started cloning.
ElHo on Mon Jan 17 10:07:02 2011 said:
By starting from a single colony one can be sure that each cell in your culture is descendend from a single founder cell, so each of your cells will have the same genetic background. If you get single colonies by plating, that's ok. I personally find it more convenient to produce single colonies by streaking, especially when not working with liquid cultures. Streaking is not difficult, you might try it. Current protocols in molecular biology was a great help to me when I started cloning.
How much is their genetic background likely to vary, anyway? And if it does, why is it a bad thing? I imagine you'd get greater reproducibility from a mixed population than you would from different clones of population?