Hi

the protocols seems OK as long as the cool down is slow

you should look at the tm of your hybrid using either the approximate formula

Tm= 2AT + 4GC

or the more accurate formula

Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) – 600/length

where [Na+] is the molar sodium concentration, (%GC) is the GC ratio, and length is the length of the sequence.

it is important to keep in mind that the length of your hybrids is 10 and the ssDNA tail should not affect the tm

if the tm is low, I would recommend to add NaCl as it will stabilise your duplex (100 to 200 mM)

I do not see the point to add the buffer and the salt after the heat

add everything (buffer + salt + DNA)

95°C for 10 min

let cool down slowly

if you have a pCR apparatus, it is something easy to program

best

T

Hi

the protocols seems OK as long as the cool down is slow

you should look at the tm of your hybrid using either the approximate formula

Tm= 2AT + 4GC

or the more accurate formula

Tm = 81.5 + 16.6(log10([Na+])) + .41*(%GC) – 600/length

where [Na+] is the molar sodium concentration, (%GC) is the GC ratio, and length is the length of the sequence.

it is important to keep in mind that the length of your hybrids is 10 and the ssDNA tail should not affect the tm

if the tm is low, I would recommend to add NaCl as it will stabilise your duplex (100 to 200 mM)

I do not see the point to add the buffer and the salt after the heat

add everything (buffer + salt + DNA)

95°C for 10 min

let cool down slowly

if you have a pCR apparatus, it is something easy to program

best

T

Here is the protocol I followed with excellent results.

Here are the compositions

Forward Oligo (First Strand)_100 picoMole------->1uL

Reverse Oligo (Second Strand)_100picoMole--->1uL

New England Biolabs Buffer 3 (NEB3)_10x------>5uL

MilliQ-------------------------------------------------------->43uL

Total Volume---------------------------------------------->50uL

Heat the reaction @95 Degrees for 5 mins and let it cool at 37 degrees for 4-5 hrs. After that keep the tube @4 degrees for 20 mins and then transfer it to -20degree refrigerator for further use.

As Thierry said there is no point adding buffer after the heat incubation. You can confirm your results by running the annealed strands on 20% PAGE against Forward (First) and Reverse (Second) strand. If you are getting band shift for your annealed product, you have got urself the same.

Here a working protocol for the lazy scientist. Put your oligos in equimolar concentration in an Eppendorf. Boil about 300 ml of water in a beaker. Put the eppendorf with the primers on a floater in the water. Leave on your bench until water is comfortable to touch, i.e. below 50oC. Done.

This is the protocole I use:

- 400ul of cacodylate solution (20mM stock solution, pH 7)

- 80ul of NaCl solution (1.5M stock solution)

- 25ul of the 1st strand solution (64uM stock solution)

- 25ul of the 2nd strand solution (64uM stock solution)

- 270ul of MilliQ water.

At the end, you have a 800ul solution with 10mM cacodylate, 150mM NaCl, 2uM of each strand. Heat it at 80ºC for 10min and let it cool down to room temperature: your double strand is formed.

The big advantage with cacodylate buffer is that the pH is not temperature-dependant!