transformed bacteria? - (Jul/06/2006 )
hello all
i have noticed strange thing in my transformed bacteria , the colonies are large ...... not the small dots as my other labmates have????
i tried this 5 times and the same colony size?
is it one colony or is it possible many colonies coalese in one????
i grow my plate for 20 hours in 37
thankx in advance
i have noticed strange thing in my transformed bacteria , the colonies are large ...... not the small dots as my other labmates have????
i tried this 5 times and the same colony size?
is it one colony or is it possible many colonies coalese in one????
i grow my plate for 20 hours in 37
thankx in advance
It's unlikely that all your clones come from colonies that have all coalesced into large colonies, unless your original cells weren't dispersed well. This is not very probable in E coli.
It could be that your construct does something to the cells to make them grow faster, and so make large colonies.
I presume your colonies have your gene of interest in them, and you can prove it.
Just how big are these monster colonies? Do they pose a threat to public safety? Should President Bush be warned of BMD (bacteria of mass distraction)? I see you're in Japan; perhaps the North Koreans were attempting to take the bugs out before they launched a pre-emtive strike against Pyongyang? Perhaps I should just have a quiet lie down for a few minutes...
As for the others in the lab, just tell them your colonies are big because you sing to them before your heat-shock step... could be fun!Seriously, sometimes the bugs just make big colonies. If it is because of your insert, at least you know it's not too toxic...
hi swanny
thankx alot for ur reply and i dont think that president Bush or anybody else should worry about it......
the problem is my labmate and my professor are growing the same cells with the same transformation protocol and even that my professor stood once beside me watching my steps and it was ok.....i dont know if i can say i sang to my cells as he was there .....
anyway yes they have my protein but still dont know why ......
about my insert i put around 100 micron suspension in the starting point and then spread.... i was thinking to centrifuge my SOC media containg the cells and take the pellet and inoculate...
thankx
How many colonies do you have compared to your labmates? Do you have more or less? When you have a lot of colonies, you will see a lot of small colonies; if you don't have so many, usually they are bigger (well, the right size). The incubation time also makes a difference because colonies will have more time to grow
I'd try increasing the selective antibiotic concentration. Eg. from 50 ug/ml ampicillin to 100 ug/ml.
I'd try also to decrease incubation time from 20 hours to 15 to 18 hours.
That'd probably work well.
Unless of course, the plate with selective antibiotic has been kept at 4degreeC for too long or the ampicillin stock has expired.
Bests,