Colony PCR or PCR with Growing culture in Broth - Trying to detect the cloned fragment in living cells (Jan/18/2007 )

I usually didnt dilute my sample.

I just pick using toothpick and chuck it in normal dry PCR tube. It gave me good results usually. Never underestimate the sensitivity of PCR.

My product is around 700bp. So I guess what perneseblue said is true about longer PCR product might be a lil hard.

Hi Timjim and perneseblue,

Thanks again for your inputs. I think everything u guys say makes much sense and I'm learning a lot even if I might not get the positive clones so fast.
I have 2 sets of cloning, a 400++bp product and the other a 900bp
Trying out with the 435bp one 1st
Tm is 57oC
I've got the PCR conditions working fine with the same insert i use for TA Cloning.

"Lastly, there is there is the possibility that there the colony PCR isn't working because there aren't any colonies which are positive."
I'm most worried about this too!!!
I'm running the gel tomorrow morning, keep u guys updated! (48samples this time)
Will pick more colonines if negative results (keeping my fingers crossed~!!!!!)

Colony PCR initiated from transformation reactions can give rise to false positives if the primers anneal to the insert sequence. (Qing Dallas-Yang, Guogiang Jiang and Frances M. Sladek, 1998. Avoiding false positives in colony PCR. BioTechniques 24 (4) 580-582) Excess DNA inserts from the ligation reaction are spread as part of the transformation reaction onto bacterial plates. Typical ligation protocols utilize 5-10 pmoles of insert PCR product in a 20 µL reaction (0.25-50 pmoles of insert/µL) with 0.5 pmoles of vector. Assuming that all the vectors acquire a single copy of insert, a minimum of 4.5-9.5 pmoles (0.225-0.475 pmole/µL) of excess insert remain in the ligation reaction. 2 µL of a 1:5 dilution of the ligation reaction (0.09-0.19 pmole) is electroporated and diluted 1:9 with SOC media (0.01-0.02 pmole/µL). If 100 µL (1-2 pmole) is spread on a 55.4 cm2 plate (0.018-0.038 pmole/mm2) and a 4 mm2 agar pick is used, then 0.072-0.152 pmoles (4.35-9.18 x 1010 molecules or approximately 2-4 pg) of background insert are available for the 100 µL colony PCR assay.
To avoid this problem during colony PCR, at least one primer should anneal to the vector. Use of the New Forward and Reverse Sequencing primers is recommended, as this allows the determination of double inserts or vector-only colonies. A negative control consisting of vector-only is also recommended for purposes of band identification. Using 10 µL of overnight culture for the screen avoids this problem.

thanks tfitzwater,

I've noted that from another thread as well and would be running with M13primers
Thanks for your reminder!

Just a note, I got a about 90% success from the morning PCR run.
Keeping fingers crossed again!!
Thanks everyone for your help!!

Wow.. 90%? that's really good! keep up the good work.

Another method u can try instead of direct PCR is to use colony blotting. Instead of picking colonies, you can just straight away screen the whole plate.

Hi Timjim,

Thanks on your suggestion, could you elaborate on colony blotting?
Would be great if I could screen the whole plate! There's many plates ahead of me to continue picking
haha

I can't really remember. But i think it something you have design probe and do a blotting to check positive colonies.