Resuspension of primers: Difference between revisions
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*Resuspend in [[TE buffer]], pH 8.0 at a concentration greater than 10μM. | *Resuspend in [[TE buffer]], pH 8.0 at a concentration greater than 10μM. | ||
*Allow to sit for 2mins, then vortex for 15s. | *Allow to sit for 2mins, then vortex for 15s. | ||
However, if you are using primers for [[PCR]]/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in [[TE buffer]] chelates Mg<sup>2+</sup> ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg<sup>2+</sup> for your reaction to proceed successfully. Each EDTA molecule chelates one Mg<sup>2+</sup> ion. | |||
Revision as of 14:21, 4 January 2006
Introduction
When receiving oligonucleotide primers from a manufacturer such as Invitrogen, the oligos arrive dry and must be resuspended in buffer. The proper choice of buffer will depend on the intended application of the primers, some common ones are:
- Sequencing/PCR
- Annealing
Method
Invitrogen recommends the following reconstitution procedure -
- Centrifuge the tube for a few seconds to get all the DNA to the bottom of the tube.
- Resuspend in TE buffer, pH 8.0 at a concentration greater than 10μM.
- Allow to sit for 2mins, then vortex for 15s.
However, if you are using primers for PCR/Sequencing you may want to resuspend in water or 100nM Tris, since the EDTA in TE buffer chelates Mg2+ ions inhibiting PCR. You could also keep track of the amount of EDTA in the mix to make sure there is still enough Mg2+ for your reaction to proceed successfully. Each EDTA molecule chelates one Mg2+ ion.
