# Optimum annealing temperature were fail with no band and just smearing - Optimum annealing temperature were fail with no band and just smearing (Jul/24/2008 )

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first of all i'm using primers commercial and designed primers

primers commercial

for the primers commercial, the annealing for the primers should be 55 degree, when i'm doing gradient PCR to determine the optimum annealing temperature, there is no band only smearing. for the gradient i'm setting 45 degree and 55 degree and i also trying 55 degree to 65 degree.

designed primers

1. for the designed primers (by me) the calculation for the primers annealing should be 57 degree, for starting i;m doing 45 - 55 degree to determine the optimum annealing temperature, the result is no band and just smearing. then the next day i'm using 55 - 65 degree and the result still the same.

2. what is difference between Primers cloning and Primers detection

p/s: the DNA genomic i was done the genomic, i don't think problem with DNA template

and my question is what happen to my "PCR product" and i had no idea to solve it

-lazyyan-

Try changing the Mg concentration.
Don't always trust the Tm calculations. If you go to three calculators, you'll get three different Tms! Use the simplest algorithm, 4 (G+C) + 2(A+T).
Try a touchdown PCR, going from 5 degrees above the calculated Tm to 5-10 degrees below it.

-swanny-

thank swanny, i will try it for next week and i'll post the result here later

-lazyyan-

my primers that's i'm using is:

Foword = 5'-ttt ggc tct ctt tta gga ct-3'

which is 4 (G+C) + 2(A+T) = 52

and;

Reserve = 5'-tag acc gta ccc tac gaa c-3'

which is 4 (G+C) + 2(A+T) = 56

today i change the concentration of pcr product as describe below:

1x time;

DNA = 3ul
Primer-F = 1 ul
primer-R = 1 ul
Mgcl = 1.5
taq = 0.5
buffer A = 2.5
dntp = 2.5
sdh20 = 8

result: the result still just have a smear,

-lazyyan-

what is the concentration of MgCl2 that you are using?

Is the genomic DNA you are using at full concentration? YOu might considered using genomic DNA that has been diluted 1:20 to 1:50 with sdwater. Using too much genomic DNA (contaminated) can result in a failed PCR reaction. Your buffer A, what kind of concentration is it? Is it 4x concentration?

-perneseblue-

the detail buffer is 10x (500mM KCL, 100mM Tris-HCL, 0.1 tritonx-100 and stabilierz,
and i'm using 1.5ul form 50nm mgcl.

should i make another dilution such 1:20 or 1:50 because before that's when i get dried pellet is just add 30ul with TE.

-lazyyan-

if the buffer is 10x concentrated, too much buffer is being used. The total volume of the formulation listed is 20ul, while the amount of 10x buffer used is 2.5ul. The total volume should be 25ul

I think the Mg ion concentration being used is a little on the high side (I assume you mean 50mM stock concentration), might want to drop it a little, perhaps to 2mM MgCl2 final (0.8ul of 50mM)

Ah yes, I would recommend diluting the template. too much DNA is also a bad thing for PCR reactions.

-perneseblue-

QUOTE (perneseblue @ Jul 28 2008, 06:46 AM)
if the buffer is 10x concentrated, too much buffer is being used. The total volume of the formulation listed is 20ul, while the amount of 10x buffer used is 2.5ul. The total volume should be 25ul

I think the Mg ion concentration being used is a little on the high side (I assume you mean 50mM stock concentration), might want to drop it a little, perhaps to 2mM MgCl2 final (0.8ul of 50mM)

Ah yes, I would recommend diluting the template. too much DNA is also a bad thing for PCR reactions.

interesting part... okey tomorrow i'll be try your advice with total volume is 25ul, which is 2.5 ul for buffer A and using 0.8 for MgCl2 and other rest for sDh20 to completed 25ul pcr product..

how do u calculated 2mM Mgcl2 be come 0.8ul

-lazyyan-

i was confusing with my taq polymerase and dNTP dilution; which is

concentration

5U/ul taq DNA polymerase
2ml x 10X Buffer A ( 500mM KCL, 100mM Tris-HCL, 0.1% Triton x-100 and stabilizers
10mM dNTP mix (quatity 0.25ml)
1ml of 50 mM MgCL2

-lazyyan-

QUOTE (lazyyan @ Jul 28 2008, 09:50 PM)

my primers that's i'm using is:

Foword = 5'-ttt ggc tct ctt tta gga ct-3'

which is 4 (G+C) + 2(A+T) = 52

and;

Reserve = 5'-tag acc gta ccc tac gaa c-3'

which is 4 (G+C) + 2(A+T) = 56

today i change the concentration of pcr product as describe below:

1x time;

DNA = 3ul
Primer-F = 1 ul
primer-R = 1 ul
Mgcl = 1.5
taq = 0.5
buffer A = 2.5
dntp = 2.5
sdh20 = 8

result: the result still just have a smear,