PCR Calculations Calculating Tm Chapter 14, SCST Training Manual 4(G + C) + 2(A + T) = Tm Manual Tm Calculation Forward Primer CCA

A TCC CTT CCT CCA AAT AGA T 4(1 + 9) + 2(6 + 6) = 64 Reverse Primer CTT CCA CAC CCT AGT TTA GTG ACA A 4(3 + 8) + 2(7 + 7) = 72

C1*V1 = C2*V2 Stock Concentration X Required Volume = Reaction Concentration X Required Volume IC*IV = FC*FV Initial Concentration X Initial Volume = Final Concentration X Final Volume

UNIT KEY mM = millimolar uL = microliter U = enzyme units Ng = nanograms

Conversions. 1 gram (g) = 1000 milligrams (mg); 1 milligram (mg) = 1000 microgram (ug); 1 microgram (ug) = 1000 nanogram (ng);

Conversion between units u will be used for the symbol for micro below mM = 1e-3 M, so 1000 mM = 1 M uM = 1e-6 M, so 1000(1000 uM) = 1 M differ by a factor of 1000 with uM smaller than mM smaller than M. To convert from one unit to another, multiply by a conversion factor equal to one. Arrange the units so that they cancel, leaving you with the desired units. To convert 0.500 M to mM: To convert 25 uM to mM

0.500 M x 1000 mM = 500 mM 25 uM x 1 mM = 0.025 mM ------------1M 1000 uM Multiply by 1000 to convert each step M to mM to uM (unit gets smaller so there are more of them in the answer) or divide by 1000 to convert each step from uM to mM to M (unit gets larger so there are less of them in the answer). The short cut is to move the decimal over three places --- remember when you have the tinier unit you will have the larger number in the value: 0.100 mM = 100 uM 45 mM = 0.045 M These examples were in units of concentration (molar: M, mM, uM) For units of distance (meters: m, mm, um, nm), volume (liters: L, mL, uL also written l, ml, ul), amount (moles: mol, mmol, umol, nmol), and mass (grams: kg, g, mg, ug), conversions are similar. Note: 1 nm = 1 nanometer= 1e-9 m 1 kg= 1 kilogram= 1000 g

Prefix kilo- (k) centi- (c) milli- (m) micro- (u) nano- (n) pico- (p)

Scientific Notation = 10^3 = 10-2 = 10-3 = 10-6 = 10-9 = 10-12

Decimal equivalents = 1000 = 0.01 = 0.001 = 0.000001 = 0.000000001 = 0.000000000001

Example Units kilogram (kg); kilometer (km) centimeter (cm) milligram (mg); millimeter (mm) microgram (ug) microliter (uL) nanogram (ng) nanoamperes (nA) picogram (pg) picoamperes (pA)

A Typical PCR Reaction --There are many variations on this theme-Taq polymerase PCR buffer (mgcl2) 1U 22mM Tris-HCl(pH8.4) 55mM KCl 1.65mM MgCl2 220mM 220mM 220mM 220mM 200nM 200nM 10-50 ng

dGTP dATP dTTP dCTP Primer 1 Primer 2 DNA template

PCR Reaction Example - the Buffer is purchased with mgcl2 Reagent Buffer (mgcl2) dNTPs Primer 1 Primer 2 Taq Water DNA Total Volume Stock Conc. 10X 10 mM 100 uM 100 uM 1 unit/ul Final Conc Required/ rxn 1X 200 uM 0.05 uM 0.05 uM 0.5 unit/rxn

= Final volume is 10.00 uL

Volume 10 ul rxn 1.00 ul 0.20 ul 0.05 ul 0.05 ul 0.50 ul 6.20 ul 2.00 ul 10.00 ul

100 ng/ul

200 ng

Thermal cycling parameters 25 35 cycles 94oC15-30 sec. 55oC15-30 sec. 72oC1min. Per kb Hold at 4oC

PCR Reaction Calculations (page 1) Reaction Components

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 10ul

Final Concentration

Reaction Components

Reaction Volume = 10ul

PCFinal Concentration 1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a.

1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 10.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

10.0 ul

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

Reaction Volume = 25ul

Final Concentration

Reaction Components

Reaction Volume = 25ul

Final Concentration

1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 25.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 25.0 ul

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 50ul

Final Concentration

Reaction Components

Reaction Volume = 50ul

Final Concentration

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a. = 50ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a. = 50ul

PCR Reaction Calculations (page 2) Reaction Components

Taq (5U/ul) MgCl2 (100mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 10ul

Final Concentration

Reaction Components

Reaction Volume = 10ul

Final Concentration

5U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 10.0 ul

Taq (5U/ul) MgCl2 (100mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

5U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 10.0 ul

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

Reaction Volume = 20 ul

Final Concentration

Reaction Components

Reaction Volume = 20 ul

Final Concentration

1U 2mM 1X 1mM 0.3uM 0.3uM 2ng/ul n.a. 20.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

1U 2mM 1X 1mM 0.3uM 0.3uM 2ng/ul n.a. 20.0 ul

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 100 ul

Final Concentration

Reaction Components

Reaction Volume = 100 ul

Final Concentration

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a. 100.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a. 100.0 ul

PCR Reaction Calculations (page 1) Answer Key Reaction Components

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 10ul

Final Concentration

Reaction Components

1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 10.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 10ul 0.2 0.4 1.0 1.0 0.03 0.03 1.0 6.34 10.0 ul

PCFinal Concentration 1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a.

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

Reaction Volume = 25ul

Final Concentration

Reaction Components

1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 25.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

Reaction Volume = 25ul 0.2 1.0 2.5 2.5 0.15 0.15 2.5 16.0 25.0 ul

Final Concentration

1U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a.

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 50ul

Final Concentration

Reaction Components

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a. = 50ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 50ul 0.2 4.0 5.0 7.5 0.5 0.5 5.0 27.3 50.0 ul

Final Concentration

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a.

PCR Reaction Calculations (page 2) Answer Key Reaction Components

Taq (5U/ul) MgCl2 (100mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 10ul

Final Concentration

Reaction Components

5U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a. 10.0 ul

Taq (5U/ul) MgCl2 (100mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 10ul 1.0 0.2 1.0 1.0 0.03 0.03 1.0 5.74 10.0 ul

Final Concentration

5U 2mM 1X 1mM 0.3uM 0.3uM 5ng/ul n.a.

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

Reaction Volume = 20 ul

Final Concentration

Reaction Components

1U 2mM 1X 1mM 0.3uM 0.3uM 2ng/ul n.a. 20.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (50mM) Primer 2 (50mM) DNA (50ng/ul) H2O Total

Reaction Volume = 20 ul 0.2 0.8 2.0 2.0 0.12 0.12 0.8 13.96 20.0 ul

Final Concentration

1U 2mM 1X 1mM 0.3uM 0.3uM 2ng/ul n.a.

Reaction Components
Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 100 ul

Final Concentration

Reaction Components

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a. 100.0 ul

Taq (5U/ul) MgCl2 (50mM) 10X buffer dNTPs (10mM) Primer 1 (100mM) Primer 2 (100mM) DNA (50ng/ul) H2O Total

Reaction Volume = 100 ul 0.2 8.0 10.0 15.0 1.0 1.0 10.0 54.8 100.0 ul

Final Concentration

1U 4mM 1X 1.5mM 1.0uM 1.0uM 5ng/ul n.a.