how to determin the Annealing Temp of ristriction sites modified primer? - help me ! (Mar/23/2006 )

Hi ladies and gentlemen:

Recently, I am busy in design primers containing ristriction sites on both 5' end of the primer. Then I encounter a problem. How to determine the Annealing Temp when PCR with the modified primer?
I just should consider the Annealing Temp of the oligos completely comlementary the template? or I should also take the restriction sites into account? Whether should I design the PCR program in the firs3-5 cylcles with a lower Annealing Temp (Annealing Temp of Oligo completely comlementary to the Template) then Improve the Annealing Temp? or just PCR with the Annealing Temp of Oligo completely comlementary to the Template? by the way, How to consider the Tm difference? I mean that If A Pair of primers selected by Oligo6.22 with a low Tm difference, when the different restriction sites are added on 5' end of forward primer and reverse primer respectfully, consequently the Tm difference will alter, If the modefied primers Tm difference is more than 5 C degree? Which should I accept? former or later?

Many thanks!

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hi

are u trying to introduce the restriction site only to the 5 'end?

if ur trying to design forward primer and reverse primer for pcr with restriction sites at each end ,

then after designing the primers
enter them in this site

http://trishul.sci.gu.edu.au/tools/OligoCalculator.html


and see if the temperature of both the primers are same

lenght , try to keep it atleast 25

then order the primers

and while doing pcr take the annealing temperature as the one u got from the site

hope it is clear and is what u asked for

all the best

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Hi phytoviridae:

Thanks for your reply!

I enter my primers in http://trishul.sci.gu.edu.au/tools/OligoCa...ml,Consequently, I got the Tm value. But unfortunately, It is different from the Tm value given by Oligo6.22. Tm value given by This site is 63c degree, and the Tm value given by Oligo is 80c degree, the former is much lower than the later. which should I accept?

I can get the Tm value of both primers completely complementary to the Template, and ristriction sites modefied primers. Annealing Temp can be roughly estimated by the Tm value.namely, Annealing Temp used in PCR= Tm +/-5c degree. now problem appeared, which Tm here should be used, Tm of oligos completely complementary to the Template, or Tm of the modefied primers?

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hi
if you add restriction sites in 5', the 15 first cycles annealing temp should be calculated without these bases that do not anneal with origial template. Then for 20 to 25 cycles, take whole primer in consideration for annealing temp.
Max temp recommended is 68° if you elongs at 72.

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Hi fred_33:

Thanks for your reply!

I will Try it. By the way I wonder, according to your experiences, what is effect of this "two-step" PCR program(in the first 15 cycles annealing temp is caculated without the bases that do not anneal with origial template. Then in later 20-25cycles annealing Temp should be improved up to 68 degree)

Yours

Albert

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I'm not trying to slam anyone's ideas, but the idea of starting with a low annealing temperature and ramping it up after the first couple cycles is contrary to a published method I use regularly with great results.

Touchdown PCR

In this method, you start high and gradually go lower, so that your early cycles amplify with higher specificity, effectively providing more accurate template for your later cycles. Using very simple primer design (I take the coding sequence, add 1 for each A/T and 2 for each G/C, and stop after I reach the number "30"), I've had about a 90% success rate with amplification via the basic program, and about another 5% on top of that can be teased out by subtly altering the temperatures.

I know, it's almost counterintuitive when you first consider it, but it really does work.

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Hi aludlam:

Thanks for your reply!

You know that my primers is modefied by ristriction sites on5‘end of both the upper primer and lower primer, the Tm of the Oligo that Anneal with the Orginal Template DNA which is lower than the Tm of the whole modefied primer. If I adopt the Touch Down PCR, My primer probably can not anneal with the Template DNA in the First couple cycles at a higher Temp. How can My PCR go?

Yours

Albert


Hope for more experiences

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Albert- I recently went through a similar experience. I had done much cloning and much PCR, but had never done PCR with added sequence at either end to add RE sites, so I learned all the lessons you are learning now

the annealing temp of my whole primers was well over 70

the annealing temp of only the initial complementary parts was about 58

so I basically did the opposite of aludlum's touchdown protocol, incorporating advice from some colleagues, as well as some of Fred's posts (he's awesome, he knows his stuff)

I started with a gradient (if you don't have access to a cycler that will do gradients you may have to troubleshoot your initial annealing temp a little). I did 10 cycles of PCR with a gradient across the block of +/- 5 degrees C from 58 (for the initial complementary parts); then I did 25 cycles of PCR with the higher Tm (in this case, I just did 94 15s / 72 90s because the Tm of the longer primers was about 75).

worked like a charm. I hope this can help you

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Hi aimikins:

Thanks fpr your feedback!

I wonder what is the result of your PCR? By the way, how do you determine the Annealing Temp of your Primers? Depend on the Software such as Oligo or DNAstar?There is a big difference between The Tm Value given by the Oligo6 and The Tm Value given by http://trishul.sci.gu.edu.au/tools/OligoCalculator.html, which should I believe? And How can I calculate the Tm value? Is there a common criterion?

Yours

Albert

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there are many ways to calculate Tm. I use a tool on Finnzyme's website because I used their polymerase. first I pasted in the parts of the primers complementary to the chromosomal DNA to get the first Tm for the first part of the amplification, then repeated with the whole primers to get the Tm for the second part

the PCR worked. I did use a gradient for the first part though; I figure Tm calculators are never 100% accurate anyways so a gradient is a good idea if you're not quite sure you're going to hit the mark and you don't want to have to repeat the run

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