How do you dilute stock primers?

This can usually be found on the tube itself or the primer sheet supplied with the order. For every 1 nmoles, add 10 μL of PCR-grade water. For example, if a primer states 19.4 nmoles, then add 194 μL of PCR-grade water. Mix the solution by vortexing to reconstitute the primers.

Why do you need to dilute stock primers?

First create a master 100 uM stock (for each primer) and then dilute it to a 10 uM working 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.

Why do we then dilute our primer stocks in water rather than TE to make our working stock for PCR?

Well, DEPC water can be acidic which may cause the oligo to degrade. TE buffer (10mM Tris: 0.1mM EDTA; pH 8.0) is the safest to dilute primers. This will ensure that the oligo pellet is at the bottom of the tube and will not be lost when the cap is opened.

How do you dilute a PCR product?

Dilution of PCR Productions

The final option is to simply dilute your PCR product in water with no further cleanup.
If you have a well optimised PCR reaction, the residual primers and dNTPs will be low and diluting may be all that is necessary.

Can I dilute primer with water?

you can use distilled water for primer dilution. Water dissolves the oligos and you can store them in -20 for long.

How do you dilute 100mm to 10mm?

See below for the dilution factor equations. For example, if a 100 mM stock solution is diluted to yield a 10 mM solution, the resulting dilution factor is 10. For this particular dilution, it can also be said that the stock solution was diluted 10-fold.

Should I dilute primer with water?

Some primers are formulated for spray application and don’t need to be thinned at all; others lose their effectiveness when thinned. If it sprays thickly or with difficulty, you do need to thin it. Add a small amount of clean water to water-based primer or a small amount of mineral spirits to oil-based primer.

Can I dilute primers with water?

Either TE buffer or nuclease-free water can be used for primer dilution. If primers are to be stored long-term, however, TE buffer may be the better choice since it can help prevent degradation.

How do you dilute Sigma primers?

Quantity Verification

Resuspend the oligonucleotide in 400 µL of water or buffer.
Dilute 12 µL into 988 µL of sterile, nuclease-free water.
Take an A260 reading of the 1 mL sample in a cuvette.
Ensure the OD value is in the linear range (~0.1 to 1 OD).
Multiply the OD of the sample volume by the dilution factor.

How much thinner do I mix with primer?

For spray applications on construction or home projects, begin by reducing the primer by 10 percent. This would call for adding approximately 3 oz. of paint thinner to each quart of primer (32 oz. x 0.10 = 3.2 oz.), or roughly 13 oz.

Should you water down primer?

Like paints, primers can be too thick to spray, and you might have to thin them slightly to get good results. Some primers are formulated for spray application and don’t need to be thinned at all; others lose their effectiveness when thinned.

How do you dilute primers for PCR?

just dilute it using NF water. Use what is recommended. Primer is dissolved in the appropriate amount of free nuclease water to reach final concentration 100 pmole as a stock solution. For work of PCR the primers then diluted to 10 pmol/µl by dilution of 10 µl of stock with 90 µl of free nuclease water.

How do you make a 10 um PCR solution?

If you want a Working Solution of 10 uM make a 1:10 dilution from the stock: Add 1.0 μL from Stock (100 μM) to 9.0 of ddH2O or TE buffer (1:10). 100 μM x 1 μL / 10 uL = 10 μM Concentration in your final PCR mix: C1V1 = C2V2

How much primer do I need for a 10 µm PCR?

– 1:10 giving a 10 µM solution for genomic PCR If you use 1 µl of this stock in a 20 µl PCR mix, your are using 0.4 µM of primer in the final mix. 9. PCR set-up using YorkBio or Promega Taq polymerase (formerly we used Bioline, now more expensive): · 1 µl 10 mM dNTPmix or 4µ1 of 2.5 mM dNTPmix (final conc. 200 µM; see below)

How do you dilute the primer and probe?

To dilute the primer and probe, use the following calculation. C 1V 1=C 2V 2 Where C 1 = Initial Concentration of solution V 1 = Initial Volume of solution C 2 = Final Concentration of solution V 2 = Final Volume of solution Solve for V 1 to calculate the volume of each stock primer needed per reaction. 50 μmols V 1 = 0.9 μmols 50 μl