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In Topic: CFU measurments
26 April 2013 - 04:33 AM
Paulgs3, on 26 April 2013 - 04:26 AM, said:
I agree there could be a ton of reasons why: viability caused by incubation time, media formulation, incubation temp, shaker speed....
A log difference in cfu is rather significant to me.
A log difference in cfu is rather significant to me.
But here: since we dont know what might have gone "wrong" or different it means pretty much nothing.
Maybe I was not clear enough about that.
In Topic: CFU measurments
25 April 2013 - 07:13 AM
Fiaq Khan, on 25 April 2013 - 04:32 AM, said:
I have done CFU measurement of E.coli at OD~0.3. I am getting different results as describe below:
First time I did the experiments the CFU results showed that 0.3 OD = 2.07 x 108 cells/ml.
After some weeks I did the same experiment and the CFU results are showed to me that 0.3 OD = 1.41 x 109 cells/ml.
I am not sure why I am getting that different values of CFU for same OD?
Any help will be highly appreciated. Thanks
First time I did the experiments the CFU results showed that 0.3 OD = 2.07 x 108 cells/ml.
After some weeks I did the same experiment and the CFU results are showed to me that 0.3 OD = 1.41 x 109 cells/ml.
I am not sure why I am getting that different values of CFU for same OD?
Any help will be highly appreciated. Thanks
It can be because of so many things... Not the same amount of cells at the start... not the same time of incubation? Less or more aeration....
Also: in the end the difference is not that big...
In Topic: Do I need to sequence after digestion?
24 April 2013 - 01:04 AM
bigudukaz, on 24 April 2013 - 12:08 AM, said:
pito, on 23 April 2013 - 10:06 AM, said:
bigudukaz, on 23 April 2013 - 12:05 AM, said:
Well i sequence only after transformation and selectivity on agar and after PCR confirmation. But i do it only, when i am sure that the positive transformation is at high chance. if you are constructing plasmid in many steps - adding few inserts, then you should sequence to make sure that your work is going forward...
I do so just because sequencing is pretty expensive as we have to use the company service and we dont have a contract with any of such companies..;.
I do so just because sequencing is pretty expensive as we have to use the company service and we dont have a contract with any of such companies..;.
The other methodes are more timeconsuming and thus often more expensive.
Well We pay for one sequence ~20 euros and to be sure - it has to overlap so its 20 euros. I preffer beeing on the safe side and do simple colony PCR to know weather there is a correct size insert and choose "better" looking (from PCR) colonies to grow->purify plasmid. Maybe its not usefull, but i was tough to do so in my lab and the reasoning was the one i just wrote
Thats a lot...
There are companies that offer plasmid sequencing for 5 dollars.
In Topic: Do I need to sequence after digestion?
23 April 2013 - 10:06 AM
bigudukaz, on 23 April 2013 - 12:05 AM, said:
Well i sequence only after transformation and selectivity on agar and after PCR confirmation. But i do it only, when i am sure that the positive transformation is at high chance. if you are constructing plasmid in many steps - adding few inserts, then you should sequence to make sure that your work is going forward...
I do so just because sequencing is pretty expensive as we have to use the company service and we dont have a contract with any of such companies..;.
I do so just because sequencing is pretty expensive as we have to use the company service and we dont have a contract with any of such companies..;.
The other methodes are more timeconsuming and thus often more expensive.
