I frequently use colony PCR to check my colonies after a cloning. It is nice
and dirty and saves you a lot of time. I usually resuspend the colonies
directly from my transformation plate in 20 ul LB each. I then use 5 ul of
that for each rxn:
5 ul bacteria resuspension
1 ul Primer1 (20 uM)
1 ul Primer2
0.5 ul 10mM dNTP
5 ul Taq buffer
0.25 ul Taq
37.25 ul H2O
I make a master mix of all the above ingredients except the bacteria, lazy
as I am. Just multipy all volumes with (the # of colonies you want to check
+ 0.5)
PCR program:
5' 96 C
30'' 96 C
30'' 55 C 25 cycles
1' 72 C pr. kb
5' 72 C
4 C
2-3 ul of that on a GIBCO minigel. I guess you can make the PCR in a smaller
volume and save some Taq buffer, if that is important to you. You would
probably have to use less bacteria then.
When you have identified your right clones, inoculate 2 ml LB+antibiotics
with the rest of the corresponding bact. resusp. Incubate as you use to, and
use that culture to inoculate a largescale plasmid prep.
In case you have re-streaked your colonies on a new LB plate already (and
then have more colonies of each clone), you can dump the colonies directly
into the PCR mix without resuspension in LB. I never re-streak, so I have to
save some bact for later plasmid prep using the LB resuspension.
Good luck
Jens
Jens Tornøe
NeuroSearch
Pederstrup, Denmark
http://www.neurosearch.com
----- Original Message -----
From: Chong Wai Yin( Zhang Weixian) <wychong at singnet.com.sg>
Newsgroups:
bionet.molbio.methds-reagnts,bionet.cellbiol,bionet.microbiology,bionet.immu
nology
Sent: Saturday, December 11, 1999 8:37 AM
Subject: colony PCR
> Hello, does anyone have experience or have a protocol in doing colony PCR?
> Thank you for helping me out.
>> Regards,
>> Kelvin
>> -- For Nature, heartless, witless Nature
> Will neither know nor care.
> And from my pillow, looking forth by light
> Of moon or favouring stars, I could behold
> The antechapel where the statue stood
> Of Newton with his prism and silent face,
> The marble index of a mind for ever
> Voyaging through strange seas of Thought, alone. --
>>
