Making blunt ends DNA with T4 Polymerase - (Nov/11/2008 )
Hi, I need to make blunt end ligation from RE digestion product and I'm using T4 polymerase from NEB. This is their protocol:
"Protocol for blunting ends by 3´ overhang removal and 3´ recessed end fill-in:
DNA should be dissolved in any 1X restriction enzyme reaction NEBuffer or 1X T4 DNA Polymerase Reaction Buffer supplemented with 100 µM dNTPs. Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes (1,2). CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme."
I did it several times in the past but without success. I don't know the 100 uM dNTPs they mentioned is the total concentration of all dNTPs or 100 uM for each of them? I just inactivate the enzyme by heating at 70°C for 15 min with 10 mM of EDTA and then purify this mixture with PCR purification Kit of QIAgen before ligation. So what could be the mistake there (I calculated the concentration needed carefully)? Does anyone have experience in doing this reaction pls suggest me, I really appreciate your support! Thank you.
"Protocol for blunting ends by 3´ overhang removal and 3´ recessed end fill-in:
DNA should be dissolved in any 1X restriction enzyme reaction NEBuffer or 1X T4 DNA Polymerase Reaction Buffer supplemented with 100 µM dNTPs. Add 1 unit T4 DNA Polymerase per microgram DNA and incubate 15 minutes at 12°C. Stop reaction by adding EDTA to a final concentration of 10 mM and heating to 75°C for 20 minutes (1,2). CAUTION: Elevated temperatures, excessive amounts of enzyme, failure to supplement with dNTPs or long reaction times may result in recessed ends due to the 3´→ 5´ exonuclease activity of the enzyme."
I did it several times in the past but without success. I don't know the 100 uM dNTPs they mentioned is the total concentration of all dNTPs or 100 uM for each of them? I just inactivate the enzyme by heating at 70°C for 15 min with 10 mM of EDTA and then purify this mixture with PCR purification Kit of QIAgen before ligation. So what could be the mistake there (I calculated the concentration needed carefully)? Does anyone have experience in doing this reaction pls suggest me, I really appreciate your support! Thank you.
When I looked at the NEB quick blunting kit protocol, it said to do the blunting reaction at room temp. Your reaction at 12 degrees was probably too low to do anything worthwhile.
I took this paragraph from NEB website, it's also written in their book of 2004 the same thing. Maybe you looked into the quick ligation kit for blunt ended DNA. What I'm doing is blunt my sticky ended DNA into blunt end.
Sorry, I don't have time right now to check this, so I'd see what the NEB website suggests. They have been known to significantly change protocols from one year to the next.
In any case, I'd still try the reaction at a higher temp. All you'll do is blunt end the DNA. You could try a time-course expt, if you're worried about potential damage, but that will just mean more samples to test later.
All the best with it.
Hello.
I just checked back to NEB website and found out that their current and 3-months-ago protocols were different...obviously they updated it 
This is my protocol, just for your reference:
digested DNA - 3ul
10x NEbuffer 2 - 2ul
10x BSA - 2ul
dNTP(5mM) - 0.8ul final conc:200uM
T4 polymerase - 0.5ul
water - (adjust to total volume 20ul)
i make it at 12oC, 20 min. Then i put it on ice and perform purification using PCR purification kit directly followed by ligation. They did not mention about adding EDTA at that time
To answer your question, i interprete 100uM dNTPs as a mixture of dNTPs which consists of 100uM dATP, 100uM dTTP, 100uM dGTP and 100uM dCTP.
Thx dcch and swanny so much, it's really helpful to me ^^!