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PCR amplification

Primer design checklist

  • Design tools:
  • Size: 15-30 nt
  • Melting temperature (Tm):
    • The temperature at which half of the oligo molecules are single-stranded (and thus "melted") and half are double-stranded (i.e., annealed to its complementary strand).
    • Depends on sequence length, composition, primer and template concentrations, salts etc.
    • Difference between Tm's of the primers should be less than 5°C.
    • Tm calculation:
    • Annealing temperature during thermocycling is set 3-5 C below Tm
  • GC content should be between 35−80% (ideally 40-60%) or equivalent to the product being amplified.
  • Delta G value of any self-dimers, hairpins, and heterodimers should be weaker (more positive) than −9 kcal/mol. Positive numbers indicate that the actual secondary structure shown will not form at all.
  • Avoid 3' complementarity between the two primers to prevent primer dimers.
  • Have your primers end with a G or C, or, if possible, CC, GG, CG, or GC in order to help the end of the primer attach better and help DNA polymerase to initiate complimentary strand synthesis. However, avoid placing more than three G or C nucleotides at the 3’-end to lower the risk of non-specific priming.

Procedure

  1. Mix the following components on ice (except for hot-start DNA polymerases) in the order listed below.
Component Input conc. Final conc. range Common final. conc. Common vol. (uL) Notes
Milli-Q water 32.5
5X Phusion™ Plus Buffer 5X 1X 1X 10 Provides 1.7 mM MgCl2
dNTPs (mM) 10 .2 .2 1
Forward primer (uM) 10 .1-1 .5 2.5
Reverse primer (uM) 10 .1-1 .5 2.5
Template DNA (uM) 1 .02 .02 1 Input amount: 5 amol - 5 fmol, or:
- 0.01 - 10 ng of plasmids
- 5 - 100 ng of synthetic fragments
5X Phusion™ GC Enhancer (optional) 5X 1X 1X For GC-rich template DNA (>65% of GC)
Phusion™ Plus DNA Polymerase 100X 1X 1X .5 Pipette gently, as the high glycerol content may lead to pipetting errors
Total 50
  1. (Optional) Control: No Phusion Plus DNA Polymerase and / or no Template DNA.
    • No or very faint DNA band should be visible on a gel. Helps to distinguish between the input material and a genuine amplification product.
    • Useful when working with a DNA sample for the first time.
  2. Run a thermal cycler program:
Stage Cycle step Temperature (°C) Time Cycles Notes
1 Initial denaturation 98 30 s - 5 min 1
2 Denaturation 98 5-10 s 25-35 Fewer cycles lead to less bias (but lower yield too) in the DNA library amplification
Annealing 50-72 10-30 s Compute based on Tm. When 72C, called "2-step PCR".
Extension 72 15-90 s
3 Final extension 72 2 min 1
Incubation 4 Hold Hold
Stage Cycle step Temperature (°C) Time Cycles
1 Initial denaturation 98 30 s 1
2 Denaturation 98 5 s 30
Annealing 60 10 s
Extension 72 30 s
3 Final extension 72 2 min 1
Incubation 4 Hold Hold

Expected yield

After 30 cycles, about 100X amplification is expected.

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