10 replies to this topic

[prabhubct]

Hello,
      I am doing colony PCR (taking 10ul culture , boiling it for 5 min and using 0.5ul for PCR)  (2kb) and trying to digest PCR product with KpnI. But it is not digesting. Is it because of Colony PCR inhibitors which may be present in PCR?. I got a smear in gel with no digestion.

[metionina]

Smear in gel for your PCR product or after digestion?
If your PCR is ok, you need to purify your PCR product before digestion.

I don't know what type of culture you use, but isn't 0.5 ul a too little volume to use for a colony PCR?

[ascacioc]

I am doing colony PCR with colonies not culture (you know the colony in colony PCR stands for...colony:) ) But I totally agree with Metionina, you need to always PCR clean-up your DNA before digestion; these enzymes are quite sensitive to different buffers. I even elute my DNA in water for digestion instead of elution buffer to keep it as clean/ion free as possible since restriction enzymes are inhibited by high salt concentrations.

Andreea

[ascacioc]

BTW: please do not post the same question two times. I have deleted the other one

[metionina]

ascacioc, on 12 September 2012 - 03:14 AM, said:

you know the colony in colony PCR stands for...colony

I thought prabhubct meant he picked a colony, made a liquid culture and used it for PCR

[ascacioc]

That's what he did. What I said should be taken as:

I save one day of waiting by directly picking the colony and put it in the PCR tube and PCR that. Like this, 0.5 uL or whatever are not too little or too much. And... you don't get your PCR inhibited by the stuff the cells secreted ON in the entire culture. Colony PCR is the PCR of a colony

Andreea

[leelee]

I've digested PCR products without cleaning them up first, and it worked for me. Not from colony PCR though.

Its probably worth just doing a clean up just to be on the safe side. But other things to consider are- how much of your digest volume is your PCR product? Can you post up your volumes?

Also, with your colony PCR- did you mean you take a stab of colony, emulsify in 10ul of water, boil and then use this for your template? Just to clarify (its the way I do it, so that's how I read your post to mean?).

[ascacioc]

@leelee: It usually works also with restriction of the PCR without clean-up, but not with all restriction enzymes. Some of them are really inhibited by high salt. +Colony PCR contains all the debris and garbage from cells growing ON. Too much salts and stuff floating around for restriction enzymes, for my taste.

How I do my colony PCR: I take a colony with the tip from the agar: this means barely touching the colony, not grabbing the entire thing + the agar underneath. Then I touch another new agar plate with this tip leaving most of the colonies there for replica plate and the rest I wash in 10 uL of the PCR master mix that is basically my PCR. You have to see it to understand it Basically I pre-dilute my colony by having the in between replica plate

[prabhubct]

leelee, on 12 September 2012 - 04:32 AM, said:

I've digested PCR products without cleaning them up first, and it worked for me. Not from colony PCR though.

Its probably worth just doing a clean up just to be on the safe side. But other things to consider are- how much of your digest volume is your PCR product? Can you post up your volumes?

Also, with your colony PCR- did you mean you take a stab of colony, emulsify in 10ul of water, boil and then use this for your template? Just to clarify (its the way I do it, so that's how I read your post to mean?).

I took 10 ul O/N culture which is grown from single colony.

[prabhubct]

ascacioc, on 12 September 2012 - 04:52 AM, said:

@leelee: It usually works also with restriction of the PCR without clean-up, but not with all restriction enzymes. Some of them are really inhibited by high salt. +Colony PCR contains all the debris and garbage from cells growing ON. Too much salts and stuff floating around for restriction enzymes, for my taste.

For KpnI digestion is possible by unpurified PCR product.http://www.fermentas...ymes/er052-kpni

[prabhubct]

ascacioc, on 12 September 2012 - 03:16 AM, said:

BTW: please do not post the same question two times. I have deleted the other one

May be some mistake, I think I may have hit post button double or something. thanks.