Hi Selena

I will bet in the first one. You do not need to grow colonies for PCR. We use to pick a colony with a sterile toothpick in 100 ul of water (or TE buffer), and then boil the suspension for 5 minutes to release DNA.

Hope it helps. Best luck

Paco

Hi Selena

I will bet in the first one. You do not need to grow colonies for PCR. We use to pick a colony with a sterile toothpick in 100 ul of water (or TE buffer), and then boil the suspension for 5 minutes to release DNA.

Hope it helps. Best luck

Paco

Hi Selena 

I agree with Francisco. All u need to do dip a tooth pick in colony(actually in our lab we grow colonies in lb agar plates and a pick will streaked on lb agar plate, the same pick(tooth pick) will be dipped in the PCR mix) and put on your PCR mix.

As well check the following thread

https://www.researchgate.net/post/Protocol_for_colony_PCR

Hi there,

I guess you are talking about bacteria colony PCR. Toothpick is definitely the best. And to make it even quicker you can also dip it directly into PCR reaction mix provided you don't put too much material in it and then save the clone on agar plate using the same toothpick.

I also agree that taking colony , adding it in water or TE and boiling of the same will work definitely in colony PCR.

Gud luck

my method: 50 ul h20, pic with a yellow tip a E. coli colony, mix with the water, them heat in the PCR machine  at 98ºC, for 10 min, them leave at 4ºc for 5 min. Centrifuge 13000g /10 min, use 2 ul for pcr (works for pcr volumes from 10ul to 50ul)

Hello, what we do in our lab is very simple and works fine for us: we pick the colony with the pipette tip make up and down movement in the PCR mix tube (not too much bateries) and then save it on an agar by just touching the agar with the tip. So you do not need to extract any genetic material before, indeed after having a first result form colony PCR (which we do not considered a final result, sometimes it is a false +) we extract and purify the plasmids of the positive colonies afterwards and repeat the same/or another PCR on clean material.

The first one works well. Some types of toothpicks seem to inhibit the PCR very strongly (I discovered this the hard way!), so I prefer to use pipette tips now.

Hi Selena, 

I usually pick a colony with a tip and place it in the PCR plate/tube that has been filled with water. Usually I put 5ul of water, and then subtract this amount from the water needed in the mastermix of the pcr. So you can do it the pcr the same plate, no need to transfer anything to a new one. 

Good luck!

Hi Selena, I have been successfully using vegetative cells as template in PCR reactions for the last 10 years by picking a colony (make sure it is pure) from a plate using a back side of a sterile loop and inoculating directly into the PCR mix. If you want to use broth culture, you have to dilute it (try 1ul of 10E+03 or 10E+04). Both colony and broth cultures produce reproducible results and identical to the isolated and purified DNA as a template, only milk culture does not. If you are looking for something loacated on a plasmid, then you have to isolate plasmids first before doing PCR.Regards.

Hi dear Helena

As Dragica said you can do both. Pick an isolate and pure colony from a plate using a back side of a sterile loop ( just touch the colony ) and inoculating directly into the PCR mix. Or you can inoculate it to LB Broth and then prepare different dilutions  (10E+03 or 10E+04 and 10E+05). Second way is better 'cause you can control your plasmid inoculated.

be successful  

I have always used the water method.  Just take a sterile toothpick and collect some of the colony.  Then swirl the toothpick in 10 uL sterile water.  Boil this in a water bath for ten minutes.  Finally add 1-10 uL of this mixture to your PCR mix (amount depends on how much of the colony you used - you don't need a lot.).  Others skip the boiling step and swirl the toothpick directly into the master mix.  I do this extra step because it helps break the cells open and release the DNA template.

I have done colony PCR several tımes. I usually take a pipette tip swipe either from a frozen stock, or a single colony. It works very well. I never diluted it in water. I use a 15ul PCR reaction volume.