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

Could you kindly tell me wht concn of DNA PCR product to use with 2U of HaeIII
Pl reply-urgent
as sometimes DNA is uncut and sometimes cut beautifully so I guess the product conc shud be adjusted
so wht wud be the optimal conc of the pdt to cut
Thanks

---

How long do you allow your digestion to run for?

And what volume and hence dilution do you cut your DNA in. Could you list the break down of components. Do you cut your DNA directly from the PCR mix? Accoding to NEB, any deviation from a low salt buffer severly drops digestion effciency. And most Taq based PCR mixes could be termed high salt. PCR mixes have a pH 8 to 9. Much more basic then the pH 7.5 of NEB buffers

Activity in NEBuffers:
NEBuffer 1: 50%
NEBuffer 2: 100%
NEBuffer 3: 25%
NEBuffer 4: 75%

---

---

Thanks Pernese
But the Restriction happens well sometimes so the enz and the pHs may not be the prob...Wht I wud like to know is if I need really thick bands say of a 15 microgram concentration in each band
5-6 bands then how much DNA should I be taking to cut initially?In a single gel I want everythin to look uniform.
I know i need to standardise but i was hopin i cud cut short on tht if anyone cud help...
thank u so much

---

The PCR product I use is sometimes 25 microliter i try to base tht on a chk gel .....there's where the prob is....sometimes assessment is going wrong

Restriction mix:

10 microliter of 10X restriction buffer
HaeIII 0.5 microliter and 9.5 microliter millipore water
HaeIII 0.5 microliter is 2U
Its from Supratherm around a year old

---

Hi,

If you are using 10 ul of 10X restriction buffer, your reaction voulume must be 100ul. Frankly I didn't understand your reaction mix.

If you are using 25 ul PCR product for digestion, how about following reaction mix:

25.0 ul PCR product + 3.0 ul 10X restriction buffer + 1.0 ul Enzyme + 1.0 ul water (total 30.0 ul)

It's good to gel purify you pcr product before using it for restriction digestion.

Hope it helps
Pravin

---

Thanks Pravin will try that and let u know ...

---

---

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 all for your kind inputs
I had been blindly followed a procedure frm a senior
From your mails i checked my work and saw that i should have been using only 1X restriction buffer however i was using too much
I have also standardised the concentration to be 5 microgram of DNA The bands come out beautifully....
Thank you all for your prompt and helpful replies...

---