Sample Preparation Guideline

          

Please prepare your samples based on the following guideline.

SAMPLE PREPARATION:

DNA CONCENTRATION and VOLUME

PLASMID DNA (<40kb in size)

100-200 ng/uL

Minimum 10 uL and 3uL per additional reaction

PCR PRODUCT

25-50 ng/uL

Minimum 10 uL and 3uL per additional reaction

DNA should be dissolved in DW or super low TE buffer (10mM Tris-HCl, pH8.0, 0.01mM EDTA). EDTA in conventional TE buffer (10mM Tris-HCl, pH8.0, 1mM EDTA) usually cause bad sequencing result.

Effect of EDTA on Sequencing reaction

EDTA is a chelating agent, and it reduces the effective concentration of free magnesium ions, which are necessary for full activity of DNA polymerases. When the molar ratio of EDTA to MgCl2 is close to 1, almost all DNA polymerase activity is inhibited.

PRIMER CONCENTRATION

5 picomole/uL

Minimum 10 uL and 2uL per additional reaction

PRIMIX RATIO

In case you send us your samples  premixed with your own primers, please refer to the following ratio.

* Plasmid DNA (~100 ng/ul) : Primer (2 pmol/ul) = 1.5 : 1

* PCR product (~25ng/ul) : Primer (2 pmol/ul) = 0.7 : 1

   The required mixture volume is 7~10 ul

UNIVERSAL PRIMERS available in MacrogenUSA