9 replies to this topic

[hianghao]

What does melting temperature of primer indicate?

In PCR, we need to determine the optimal annealing temperature, which is ~5C lower than Tm. I am running RACE using SMARTer RACE kit which require primers with Tm of >70C. I have a set of primer design using primer 3. The calculated Tm is 76C (Tm=4(G+C)+2(A+T)). However when i buy it the label on the primer says that the Tm is 68C. That manufacturer use another model to calculate Tm.

Now, generally Ta is around 5C lower than Tm. Which Tm should i use? I've tried Ta of 65C and 68 using normal pcr and it had terrible smear. Should i increase or decrease the Ta? I am aware that doing gradient is the best solution but i don't have a gradient cycler and i have 10 samples using different set of primers. Any tips to predict the range of Ta?

Edited by hianghao, 07 October 2010 - 06:06 AM.

[Ameya P]

If you have gotten a smear, then increase annealing temperature to get specific bands. I have used reaction conditions wherein the Ta was 70C. So my guess is that your TA is in the range of 68-74C. Since, you dont have a gradient run a reaction at 70C, if there is too much smearing, goto 72 else, move up or below as required.

[hianghao]

gt_ameya, on 07 October 2010 - 03:53 AM, said:

If you have gotten a smear, then increase annealing temperature to get specific bands. I have used reaction conditions wherein the Ta was 70C. So my guess is that your TA is in the range of 68-74C. Since, you dont have a gradient run a reaction at 70C, if there is too much smearing, goto 72 else, move up or below as required.

What does a melting temperature indicate?

I've ran 3 temperature on 3 different samples: 1,2,3; each sample with different set of primer. Gel i got were





Which sample should i increase and which should i decrease the Ta? Could the smear caused by degraded RNA/ low quality cDNA?

Edited by hianghao, 07 October 2010 - 08:00 AM.

[phage434]

I'm guessing that your problem is massively too much template in your PCR reaction.  I'd try again with 100x and 1000x less template in the reaction.  The fact that you can see long DNA fragments on your gel indicates there's far too much.

[Ameya P]

hianghao, on 07 October 2010 - 06:35 AM, said:

What does a melting temperature indicate?

The definition can be found anywhere online. But honestly, I do not understand the concept. It would be nice if somebody explained what it actually means.  

What I am thinking is, why is your ladder also smeared???? What concentration of agarose are you using? How old is the buffer in the tank?

[hianghao]

gt_ameya, on 07 October 2010 - 10:05 PM, said:

hianghao, on 07 October 2010 - 06:35 AM, said:

What does a melting temperature indicate?

The definition can be found anywhere online. But honestly, I do not understand the concept. It would be nice if somebody explained what it actually means.  

What I am thinking is, why is your ladder also smeared???? What concentration of agarose are you using? How old is the buffer in the tank?

Primer Melting Temperature ™ by definition is the temperature at which one half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. But i just don't understand.

My buffer is 2 weeks old and i used 2ul ladder, 1% agarose. Time to change buffer! That 100bp ladder is a bit degraded because when i use it side by side with 1kb ladder, the 1kb ladder was clear.

[phage434]

Good point about how bad your ladder looks.  I'd recommend working on making decent gels and good looking ladders first, then worry about your PCR.  Is the ladder lane overloaded?  Are you making your gel with water instead of buffer? Two weeks sounds like too long for buffer to last.

[hianghao]

phage434, on 08 October 2010 - 06:42 PM, said:

Good point about how bad your ladder looks.  I'd recommend working on making decent gels and good looking ladders first, then worry about your PCR.  Is the ladder lane overloaded?  Are you making your gel with water instead of buffer? Two weeks sounds like too long for buffer to last.

How frequently should i change the buffer? I used TBE to make the gel.
I ran new pcr and here's the result. This time i use 1kb marker

Ta used were 68 and 70C. Does it indicate optimal annealing? Because when i use 72C again today, there were smears. My primers Tm are 68C, 64C.I am confused because i thought Ta lower than optimised Ta will produce smear. But in my case, 65C: smear; 68,70C: band-like; 72C: smear again. Is this normal?

[Ameya P]

Ta used were 68 and 70C. Does it indicate optimal annealing? Because when i use 72C again today, there were smears. My primers Tm are 68C, 64C.I am confused because i thought Ta lower than optimised Ta will produce smear. But in my case, 65C: smear; 68,70C: band-like; 72C: smear again. Is this normal?
[/quote]

If there is some banding pattern around 68-70, then play in the region. I would suggest increasing Mg+2 concentration. It is unfortunate that you do not have a gradient thermal cycler, but what you could do is set up your reactions at one time. Make triplet sets of Mg+2 concentrations of 1, 2 and 3 uM and run then at 68, 69 and 70C. one after the other. So you have Mg+2 conc. of 1, 2 and 3 at 68C, then 69 and 70C resp. Run them all on a single gel and compare.

Secondly, it could be that your PCR conditions are not optimal as well. What conditions are you using, what Polymerase and what is the expected amplicon size. (Source of template DNA could also be helpful)

have a happy weekend....  

[hianghao]

[/quote]

If there is some banding pattern around 68-70, then play in the region. I would suggest increasing Mg+2 concentration. It is unfortunate that you do not have a gradient thermal cycler, but what you could do is set up your reactions at one time. Make triplet sets of Mg+2 concentrations of 1, 2 and 3 uM and run then at 68, 69 and 70C. one after the other. So you have Mg+2 conc. of 1, 2 and 3 at 68C, then 69 and 70C resp. Run them all on a single gel and compare.

Secondly, it could be that your PCR conditions are not optimal as well. What conditions are you using, what Polymerase and what is the expected amplicon size. (Source of template DNA could also be helpful)

have a happy weekend....  
[/quote]

I am using Advantage2 Polymerase Mix which contains 3.5mM MgCl2. Hence its not possible to reduce the conc of MgCl2. Can increase it though... Need to use that polymerase because i am running RACE protocol which require me to use that for optimal result. I don't know the length of my amplicon, but it should be less than 1kb.
I am using the protocol described in my kit:
94C 30s
Ta 30s
72C 3m
25 cycles. If the band is too faint, increase another 5 cycles.

Thanks for the advise. I'll try to increase MgCl2 conc and try for temp around that range.