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Primer Purification

Once you have the oligonucleotides from the core you will need
to process them before phosphorylation.
The first step is a quick purification:
1. Dissolve the lyophilizate provided by the Biomedical Research Core Facility in 200 uL TE buffer.
2. Add 10 uL Na Acetate (3M, pH=5.2).
3. Add 400 uL ice-cold 100% EtOH.
4. Place in dry ice for 1 hour.
5. Centrifuge @ 4C for 30 minutes.
6. Remove supernatant via aspiration.
7. Dissolve the pellet in 100 uL TE.

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Concentration Adjustment

Now that you have the purified oligo, you need to adjust its
concentration to [1 ug/uL]. To do this, take an optical density
measurement at 260 nm. 1 OD unit for single-stranded DNA @ 260 nm
is the equivalent of a 33 ug/mL.
1. Add 998 uL of ddH2O to an eppendorf tube.
2. Add 2 uL of the purified oligo to this eppendorf and mix.
3. Take an OD260 measurment of this solution.
4. Multiply this value by 16.5 to get the concentration of the
purified oligo in ug/uL.
5. Add ddH2O to adjust the oligo concentration to 1 ug/uL.

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Phosphorylation

Now that you have 1 ug/uL oligo, you can phosphorylate:
1. To an eppendorf tube add the following:
a. 35 uL ddH2O
b. 5 uL 10X T4 Polynucleotide Kinase buffer
c. 5 uL 10mM ATP solution
d. 2.5 uL Oligonucleotide (1 ug/uL)
e. 2.5 uL T4 Polynucleotide Kinase (10 U/uL)
2. Mix contents, centrifuge briefly
3. Incubate @ 37C, 1 hour.
4. Centrifuge briefly and store at -20C until used for the Ligation Step.
Note 2

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