2 | ABE PCR Guide—for program site staff
Colony Polymerase Chain Reaction Lab Protocol
Program Site Guide and Prep Guidelines
Colony PCR is a quick procedure to identify which if any transformed bacterial colonies carry the gene of interest. The traditional way this determination is made is by sub-culturing colonies growing on a Petri plate. Plasmids, purified from these overnight cultures, are then digested, and the resulting fragments run on gels. The process takes several days. Colony PCR allows the researcher to take samples of transformed cells and, in just a few hours, identify which colonies carry the gene of interest.
PCR Primers and Primer Binding Sites
This PCR procedure uses the forward and reverse primers that flank the BamHI and HindIII restriction enzyme sites in the pARA plasmid.
Forward plasmid / 5’-TGTAACAAAGCGGGACCAAAGC-3’Reverse plasmid / 5’-GCGTTTCACTTCTGAGTTCGGC-3’
The forward primer binds to positions 1,103–1,124 of the pARA and pARA-R plasmids. The reverse primer binds to positions 1,764–1,743 of the pARA and 2,194–2,173 of the pARA-R plasmid. Both primers are 22 base pairs long, have a GC content of about 50% and a Tm of about 58°C.
The position of the primer binding sites and the size of the amplified fragments are shown for both pARA (Figure 1) and pARA-R (Figure 2).
Figure 1: The diagram indicates features of the pARA plasmid including the BamHI and HindIII restriction enzyme sites, the primer binding sites, the beta-lactamase gene encoding the protein for ampicillin resistance, and araC gene encoding the AraC protein that inhibits the expression of the rfp gene in the absence of arabinose.
Figure 2: The diagram indicates the features of the pARA-R plasmid including the BamHI and HindIII restriction enzyme sites, the primer binding sites, the beta-lactamase gene encoding the protein for ampicillin resistance, the araC gene encoding the AraC protein that inhibits expression of the rfp gene in the absence of arabinose, the pBAD promoter that the AraC protein binds to, and the rfp gene encoding the red fluorescent protein.
Materials and Technician Prep Guidelines
There are only two consumables used in the PCR procedures that have not been used in the ABE program before. Purchasing details for these are shown in the table below.
Consumable / Company / Code / Cost2X PCR master mix / NEB / M0270L
Taq 2X master mix: 500 reactions / $224.00 (supplied for free along with other donations)
Primers* / Invitrogen / Custom oligonucleotides / $11.00 forward primer
$11.00 reverse primer
To order primers:
· Go to Invitrogen’s Custom DNA Oligos website
· Select “Single” Standard Oligo
· Order first oligo
o Oligo name: pARA forward
o Oligo sequence 5’ to 3’: T G T A A C A A A G C G G G A C C A A A G C
o Synthesis scale: 50 nmole
o Purification: desalted
o No modifications
o No alternate oligos
o No special handling options
o Add to cart
· Order second oligo
o Oligo name: pARA reverse
o Oligo sequence 5’ to 3’: G C G T T T C A C T T C T G A G T T C G G C
o Synthesis scale: 50 nmole
o Purification: desalted
o No modifications
o No alternate oligos
o No special handling options
o Add to cart
· Go to ‘my cart’ for shipping and payment
Material Preparation:
Prepare Master Mix (MM) according to amounts from the Colony PCR Reagents spreadsheet. To account for pipetting errors, increase the number of groups by 1 for every 12 groups requested by a teacher. For example: If a teacher requests Colony PCR for 12 groups, then make enough MM for 13 groups. If a teacher requests 24 groups, then make enough MM for 26 groups. The Taq can be added to the MM and frozen for at least three weeks prior to use.
For remaining reagents and supplies, use the following table to calculate how much of each you need to supply.
Reagent/supply / Amount needed /group / Aliquot this amount/groupp-ARA at 0.025 ng/μL / 2 / 3
p-ARA-R at 0.025 ng/μL / 2 / 3
Master Mix (MM) + Taq / 92 / 96
1 kb DNA ladder / 8 / 9
50XGRLD or 50XGGLD[i] / 10 / 14
PCR tubes / 4 / 4
1.5 mL microcentrifuge tubes / 5 / 8
p-20 tips / 15 / 20
Agarose / 0.12 g / 0.3 g
20X SB buffer / 16.5 mL / 20 mL
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[i] For gel boxes that use GelRed Loading Dye (e.g., Fotodyne), refer to the recipe “Preparation of 50XGRLD”; for MiniOne gel boxes, refer to the recipe “Preparation of 50XGGLD”.