Resuspending PCR primers and other oligos

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 Primers and DNA oligos are often received in a lyophilized state.

Primer dilution:

For primers first make a master 100 uM stock (for each primer) and then dilute it to a 10 uM working stock. Companies provide product information sheet with primes where it will be mentioned how much water should be added to get 100 uM stock.
(#this reduces the number of freeze/thaw cycles that the master primer stock goes through and reduces the chances of contaminating the primary source for the primer.)

Procedure

·        Spin Down Tubes for a brief time (1 min at Mix speed) #Primers should always be spun down before opening the tube for the first time. The pellet can become statically charged and, without this step, can either fly out of the tube or remain in the cap, resulting in loss of yield.

·    Prepare Master Stock, 100 µM b adding suggested the amount of water to the lyophilized primer (original primer tubes are used for this 100 µM stock.) Allow to sit it for 10 to 20 min at room temperature.

·         Mix well before making working stock dilutions. (vortex and spin down )

·     Prepare the working stock by diluting 1:10 from master stock. (Lable with name con. and date.)(# Primers can be stored at -20°C for years )

G-blocks and other DNA oligos:

·        Before opening the tube, spin it down in a microcentrifuge for 1 min to ensure the DNA is in the bottom of the tube.

·     Add molecular grade water, or a buffer to reach a final concentration of 10 ng/µL. (storage concentrations <1 ng/µL result in loss of material due to adherence to the plastic tube in the absence of a carrier such as tRNA.)

·         Vortex briefly.

·      Incubate at approximately 50°C for 15–20 min. (heating the tube will ensure the solvent comes in contact with the tiny pellet, even if it is stuck to the side of the tube. Thus, this step will increase the likelihood that the entire pellet will be resuspended.)

·        Briefly vortex and centrifuge.

·       Verify the final concentration. store at -20°C till further use.

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