DNA concentrations and restriction

As, exploresci stated, I too don't understand the digestion mix formulation.

If the formulation is truly

10ul Buffer (10X)
0.5ul Enzyme
9.5ul water

and of that 20ul solution (now 5x), 2ul was added to 25ul PCR mix, it would mean that the buffer concentration in the digestion mix is only 0.4X . Thus in situation where the volume of PCR mix used was smaller, the buffer concentration would approach a more optimum 1X buffer concentration. And thus cut faster, or perhaps start cutting if the buffer concentration was so suboptimal that the enzyme activity was inhibited.

I also humbly disagree with the accessment that pH is not a factor. pH will effect the cutting efficiency of the enzyme and its denaturing rate.

On another note, an enyme will cut DNA until every last recognition site has been cut. Thus even 1 U of enzyme will cut even 1mg of DNA given sufficient time. So rather then calibrate the DNA to the enzyme, one would simply give the enzyme more time to cut.

15ug DNA is alot of DNA, how about using 10U and leave it to cut overnight.

As for obtaining uniform DNA in every band, unfortunate the only way to do so is to calibrate the DNA concentration first, either by nanodrop (spectronometre) or by running a small sample on the gel.

QUOTE (perneseblue @ Sep 23 2006, 04:25 PM)

As, exploresci stated, I too don't understand the digestion mix formulation.

If the formulation is truly

10ul Buffer (10X)
0.5ul Enzyme
9.5ul water

and of that 20ul solution (now 5x), 2ul was added to 25ul PCR mix, it would mean that the buffer concentration in the digestion mix is only 0.4X . Thus in situation where the volume of PCR mix used was smaller, the buffer concentration would approach a more optimum 1X buffer concentration. And thus cut faster, or perhaps start cutting if the buffer concentration was so suboptimal that the enzyme activity was inhibited.

I also humbly disagree with the accessment that pH is not a factor. pH will effect the cutting efficiency of the enzyme and its denaturing rate.

On another note, an enyme will cut DNA until every last recognition site has been cut. Thus even 1 U of enzyme will cut even 1mg of DNA given sufficient time. So rather then calibrate the DNA to the enzyme, one would simply give the enzyme more time to cut.

15ug DNA is alot of DNA, how about using 10U and leave it to cut overnight.

As for obtaining uniform DNA in every band, unfortunate the only way to do so is to calibrate the DNA concentration first, either by nanodrop (spectronometre) or by running a small sample on the gel.

Thank you Pernese
will look into tht

One more doubt
When a procedure says to add 2U of enz to a PCR pdt.
Does it mean that we add 2U to a particular conc of DNA ....or the whole thing ie 50 microliter.
Coz if that be the case I am diluting 20U to 2U in a separate tube and adding 2U to each amplicon..so wont the 2U get diluted in 20 microliter (or whatever amplicon vol I am using) further?
Though i have standardised i wud like to know for future purpose.

Also so everytime i take a particular vol of DNA to give me 5 microgram....does it mean tht i have to adjust restrictn buffer concn to 1 X for tht volume for each sample?

QUOTE (microcharm @ Sep 27 2006, 05:40 AM)

One more doubt
When a procedure says to add 2U of enz to a PCR pdt.
Does it mean that we add 2U to a particular conc of DNA ....or the whole thing ie 50 microliter.
Coz if that be the case I am diluting 20U to 2U in a separate tube and adding 2U to each amplicon..so wont the 2U get diluted in 20 microliter (or whatever amplicon vol I am using) further?
Though i have standardised i wud like to know for future purpose.

Usually when a protocol say add 2U of enzyme to a sample, it actually means "When you add 2U of restrction enzyme A, to Xgram of DNA, all the DNA will be cleaved in Y amount of time."

Generally you need to know rough how much DNA is present and how long you are willing to wait for the reaction to complete, to decide how much enzyme to use.

With the exception of very dilute DNA, it is not the concentration of DNA, but rather the amount of DNA being cut and the time the sample is left to cut.

QUOTE (microcharm @ Sep 27 2006, 05:40 AM)

Also so everytime i take a particular vol of DNA to give me 5 microgram....does it mean tht i have to adjust restrictn buffer concn to 1 X for tht volume for each sample?

Ah, yes. So do. However I would adjust the sample volume with water, so I won’t have to adjust the restriction buffer mix for each sample.

Example
3 DNA sample, each having 5micrograme DNA but have different volumes
S1 5ug DNA 5ul
S2 5ug DNA 20ul
S3 5ug DNA 12ul

I would adjust the volume of all the reaction mixes to 20ul by adding sterile distilled water. And then add the same volume of restriction digest mix to all three (calibrated to 20ul)

DNA concentrations and restriction - restriction (Sep/21/2006 )