Primer information sent from a company will list the molecular weight of your primer (g/mol) and the amount ofnmolesthat they sent. If you have 29.4nmolof primer and you want to make a 100μMstock solution inTE– simply multiply the amount ofnmolesby a factor of ten and add that manymicroliters. In this example add 294μL.
Calculations
100μM= 100μmol/L= 0.1nmol/μL
29.4nmolX 1μL/0.1nmol= 294.0μL
Our general practice is to make a 100μMstock solution withTEand then dilute to a 10-20μMworking solution usingTrispH 8.3 or filteredddH2O.
Confirm the concentration using thenanodrop, because the concentrations provided by the company are often wrong. Tonanodropa primer you must change the setting parameters to measure a single stranded template. You will also see DNA-33, which refers to the wavelength-dependent extinction coefficient inng-cm/microliter:
• Double-stranded DNA: 50
• Single-stranded DNA: 33
• RNA: 40
Thenanodropgives readings inng/µl, but you often need to convert this value intopmol/µl. If your primer has a molecular weight of 6000g/mol, this is the same as saying 6000ng/nmol, and thenanodropreading is 9.6ng/ul, here is how to convert the valuetopmol/µl:
9.6ng/µl÷6000ng/nmol= 0.0016nmol/µl
0.0016nmol/µl X1X103pmol/nmol=1.6pmol/µ
Nucleotide concentrations
Nucleotides forPCRreactions are abbreviated asdNTPs, where theNrefers to the fournuleotides(ATCG). They are usually shipped with eachdNTPconcentrated at 100mM. They are suspended and diluted in filteredddH2O. A standardPCRreaction uses 2mMof eachdNTP.
Calculations
To make working stocks of 100μL:
M1V1=M2V2
100mMXV1= 2mMX 100μL
V1= 2μL
Therefore, add 2μLof eachdNTP(2μLX 4dNTPs= 8μL) into 92μLof filteredddH2O.
FAQs
Concentrations and Primer Resuspension? ›
Your primers will arrive as a lyophilized film at the bottom of a cryo-tube. To use them, you must resuspend them in autoclaved dH2O. Make a high-concentration stock by resuspending the lyophilized primer to a standard 100 µM concentration (that's micromolar = µmol/L = pmol/µl).
How do you resuspend PCR primers? ›To determine the amount of water to add to the lyophilized primer simply multiply the number of nmol of primer in the tube by 10. That will be the amount of water to add to make a 100 µM primer stock. For example, if there are 38.2 nmol of primer a 100 µM primer stock is created by adding 382 µl of water.
What is the concentration of primers? ›First, identify the concentration required for your working stock. For primers, this typically ranges from 10–100 µM, and for probes, from 2–10 µM.
What is the formula for primer resuspension? ›The most useful formula you will be using is: M1 V1 = M2 V2, Starting molarity (M1) multiplied by starting volume (V1) equals the desired molarity (M2) multiplied by the desired volume (V2).
What water do you use to Resuspend primers? ›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.
What concentration of primer should I use for PCR? ›The recommended primer concentration for standard PCR applications is between 0.1μM and 1μM of each primer. Generally, using a low primer concentration is better. It ensures a lower background and cleaner product.
What do you resuspend DNA in? ›Although double-stranded DNAs are stable under most standard storage conditions, we recommend the following to maintain high quality DNA: Upon receipt, briefly centrifuge tube or plate and resuspend the DNA in nuclease free Tris-EDTA (TE) buffer, pH 8.0 or 10 mM Tris-HCl, pH 8.0 to the desired concentration.
How does primer concentration affect PCR? ›Higher primer concentrations often contribute to mispriming and nonspecific amplification. On the other hand, low primer concentrations can result in low or no amplification of the desired target (Figure 3).
What is the concentration of primers in qPCR? ›At what concentration should I use my primers in my qPCR? Typical concentrations are 500 nM of each primer in the reaction.
What if primer concentration is too high in PCR? ›Using an excessive concentration of primers can increase the chance of primers binding nonspecifically to undesired sites on the template or to each other. Use well-designed primers at 0.2–1 μM in the final reaction. In addition, verify that the correct concentration was supplied by the manufacturer.
How long does it take to Resuspend primers? ›
Method. 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.
What is the reverse primer for PCR? ›Two primers are utilized, one for each of the complementary single strands of DNA released during denaturation. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand).
What is primer in PCR reaction? ›Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases.
Is it better to resuspend DNA in TE or water? ›Regardless of the storage temperature, resuspension in TE buffer, rather than in water or dried down, is best for long-term storage. Storing oligos with fluorophores. Typically, fluorophore-labeled oligos show a reduction in signal of about 20% after 24 months when stored at –20°C in TE.
Do I need to water down primer? ›Can the primer be diluted? If it is on the thick side, you should dilute it to ensure effortless application with your paint sprayer. Then pour the primer into the paint container.
How do you dilute primers for sequencing? ›How To Dilute Your Primers. If you have a primer in solution that is 100 micromolar, that is the same as 100 nmol/ml or 100 pmol/µl. We only need 12 picomoles in one sequencing reaction so dilute in the following method: Do a 1:10 dilution, take 10 µl of 100 µM solution add 90 µl of water.
How do you reverse a primer in a sequence? ›For a reverse primer: write the complement sequence of the 3' end of the sense template, reverse it, so it can be read as 5'-3' and add any extra sequence at the 5'end of this primer. Thus, for the example given above, the 5'-3' mode of the reverse primer will be: 5'- NNNNNNNNNN-CTCTAGAATCCTCAA-3'.
Can primers be reused in PCR? ›Creating the appropriate number of primers for each kit is essential for ensuring that students do not attempt to "reuse" primers and thus break them off from synthesized PCR products.
How do you mix forward and reverse primers? ›Mix together 800uL Nuclease-Free Water 100uL forward primer and 100ul reverse primer for a final working stock of 10uM forward and reverse mixture. You will use this mixture at a final concentration of 0.4uM for qPCR.