Berglund: making rNTP

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Emily Goers, Fall 2006
rNTP protocol

Final concentration of each: 100 mM
final pH: 8.1
1) Each rNTP has a slightly different mass (see below), so first calculate how much liquid (water plus acid/base) you will need to add to get a final concentration of 100 mM. Use 100mg of each rNTP for this calculation.
U 550 g/mol
C 527.1 g/mol
G 523 g/mol
A 551.1 g/mol

I recommend adding a much smaller amount of water than you will actually need, then pH using acid or base, and then adding in how much extra water you need to make it up to the final volume. However you have to watch out to not add too much water after you have pH’d because then the pH might change.
EXAMPLE: the last time I did this (5-9-06), this is roughly how much acid and/or base I used to get to the proper pH of 8.1. Generally you will only need to add base, but add it in very small quantities (uL’s) so as not to over-shoot the pH. pH using strips. Also, compare the pH of the rNTP’s to some other solutions in lab that are at pH 8. The pH strips can be misleading.

U 1820 uL final volume need for 100 mM. First added 1750 uL of water then ~34 uL 4 N NaOH, added water up to 1820 uL final volume.
A 1810 uL final volume, added ~90 uL 4N NaOH
G 1910 uL final volume, added ~31 uL 4 N NaOH
C 1890 uL final volume, added ~80 uL 4 N NaOH and 21 uL HCl

Keep the rNTPs at room temperature for as brief a period of time as possible, they aren’t very stable. Aliquot into 50-100 uL aliquots and immediately put into a –20 degrees C. Label each tube with color-coded stickers with the nucleotide. Label the outside of the main “rNTP” box with the date you made them.