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Homemade RCA (Rolling Circle Amplification) mix
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Buy:
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NEB M0269L
phi29 DNA Polymerase
1X phi29 DNA Polymerase Reaction Buffer
1X NEB rCutSmart Buffer
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NEB M0361S
Pyrophosphatase, Inorganic (E. coli)
50 mM Tris-HCl (pH 7.5)
50 mM Potassium acetate
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NEB N0447L
10mM dNTPs
10 mM MgCl2
20 mM Tris acetate (pH 7.9)
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Qiagen 19086
Buffer EB
10 mM (NH4)2SO4
10 mM Magnesium acetate
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IDT Custom DNA Oligonucleotide
Random Hexamers with two 3'-terminal phosphorothioate linkages (NNNN*N*N) 250nmol; resuspend to 100uM concentration
4 mM DTT
0.1 mg/ml Recombinant albumin
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Reaction Mix (per sample)
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0.6ul
phi29 DNA Polymerase (6 units, 10 units/ul)
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0.6ul
Pyrophosphatase, Inorganic (E. coli) (0.06 units, 0.1 units/ul)
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1ul
10 mM dNTP (1mM final concentration in 10ul)
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0.25ul
10X phi29 DNA Polymerase Reaction Buffer
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0.25ul
10X rCutSmart Buffer
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2.5ul
100uM random primers (25uM final concentration in 10ul)
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5.2ul total volume
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NB: Creating 5.2ul per sample provides extra dead volume to enable pipetting 5ul from a master mix
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Protocol
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To 5ul Buffer EB, add either 1ul DNA plasmid or pick a bacterial colony into this volume and mix well
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Heat at 95'C for 3mins
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Add 5ul Reaction Mix; do not vortex or mix
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Incubate at 37'C for >1h
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Submit for Sanger sequencing or whole-plasmid sequencing
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Cost savings
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TempliPhi DNA Amplification Kit (500 reactions)
$1,600
$3.14/reaction
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Homemade RCA (Rolling Circle Amplification) mix
$1.31/reaction
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Hence the Homemade RCA mix comes in at 2.4-fold cheaper than its commercial alternative.
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I have been using my own Homemade RCA mix for the past two months (having used the commercial alternative for the previous four years), and can confirm that it yields comparable results from Sanger sequencing from picked bacterial colonies, or purified plasmid DNA amplified by RCA.
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Note that the Homemade RCA mix seems to give poorer results when picked bacterial colonies are sent for whole-plasmid sequencing (using services such as Primordium or Plasmidsaurus) compared to its commercial alternative; however the results are comparable when instead submitting purified plasmid DNA amplified by RCA.
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