Method: 20mL Emulsion PCR (ePCR) Setup for Oligopaints (Part 1 of 3)
By Nicholas Apostolopoulos
Principle: We u a modified emulsion PCR protocol (Williams et al., Nature Methods
3(7):545-50, 2006) to amplify our oligonucleotide libraries. In this strategy, PCR master mix is beaten into a water-in-oil emulsion, with each oil droplet receiving on average 1-2 template molecules. Thus, a complex library can be amplified in a great many small, parallel reactions rather than one large pooled reaction. This approach is designed to limit the effects of template competition, template bridging, and intermolecular recombination that can distort and skew the complexity of the library during exponential amplification.
The following protocol outlines how to tup 8 x 20mL ePCR reactions done in bags optimized for u in KBiosciences HC-16 Hydrocycler. Note that the reactions could also be cycled in a Peltier thermal cycler (e.g. MJ Rearch/BioRad machines) in 96-well plates.
Time Required:
Setting up the emulsion mixture can be done within 1-2 hours. The PCR itlf takes ~4 hours, and can
be stored overnight at 4 ºC if you wish to break the emulsion the next day.
Reagents:
•ABIL EM 90, a surfactant (Degussa)
•Bovine rum albumin (BSA) (for molecular biology, powder; EMD)
•Deoxynucleoside triphosphate (dNTP) mix (ABI)
•Mineral oil (for molecular biology, light oil; Sigma-Aldrich; # M5904)
•KAPA Taq (Kapa Biosystems)
•Triton X-100, a surfactant (general-purpo grade; Fisher Scientific)
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Equipment:
•Hydrocycler (KBiosciences/LGC Genomics; 16 plate Thermal Cycler)
•Thermal Bag Sealer
•Plastic bags (KBiosciences; KBS-0004-007)
•Glass Bottle (500ml, round bottoms; Pyrex)
•Magnetic stirrer with speed controller
•Stir bar (3.7 x 0.8 cm, pivot rings, polytetrafluoroethylene; VWR International) Preparation:
Before tting up your aqueous PCR mix, you should first place your 500mL glass bottle in 4ºC cold room, and let it chill. It is very important that the emulsion is t up in the cold room, and kept cold until placed on the hydrocycler.
Procedure:
1. Prepare the oil-surfactant mixture by adding the following components to your
500mL chilled glass bottle in the cold room
Component Final concentration Vol. [134.4mL]
ABIL EM90 4% (v/v) 5.4 mL
Triton X-100 0.05% (v/v) 67.2 µl
Mineral Oil 96% (v/v) 129 mL
2. Add the
3.7 x 0.8 cm stir bar to the glass bottle, and begin stirring oil-surfactant
mixture at 1,500 r.p.m on a magnetic stir plate in cold room. Allow mixture to stir for at least 30min before adding aqueous pha and creating emulsion.
3. Meanwhile, prepare aqueous pha for the emulsion by mixing the following
components in a 50mL conical tube:
Reagents
1X 2.8 mL aqueous ePCR 16X ePCR Mix
280 µL 10X KAPA Buffer 4480 µL 10X KAPA Buffer
56 µL 10mM dNTPs 896 µL 10mM dNTPs
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14 µL Primer Forward (100µM) 224 µL Primer Forward (100µM)
14 µL Primer Rever (100µM) 224 µL Primer Rever (200µM)
56 µL Template (10ng/µL) 448 µL Template (10ng/µL)
140 µL BSA (10mg/mL) 2240 µL BSA (10mg/mL)
14 µL KAPA Taq 224 µL KAPA Taq
2233 µL dH2O 36064 µL dH2O
2800 µL ePCR aqueous mix 44800 µL ePCR aqueous mix
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Thaw all components on ice, and keep aqueous mixture on ice at all times.
Note:It is best to keep fluorescently labeled primer in the dark at all times in order to maintain the integrity of the fluorophore. For the rest of the protocol, try to minimize exposure to light.
4. Invert conical tube 6-7 times to ensure aqueous pha is properly mixed.
自己在家洗羽绒服正确洗涤方法Afterwards, aliquot 44.8mL dropwi to oil-surfactant mixture in cold room while oil pha is still stirring. After addition is complete, continue to stir for an
additional 15 minutes to create emulsion.
Note: To ensure that emulsion is properly made, you can take small aliquots of w/o emulsion and place on a microscope slide and look at aqueous pha droplets using
bright field microscopy. Droplets should range from 8 – 10 µm in size. If you find that droplets are either too small or too large, you can adjust stirring time and speed to create droplets of desired size.
5. Once emulsion is created, aliquot 20mL per al-able ePCR bag. Place bags on
ice and cover.
6. Once bags are filled, al top of bags using adhesive strip, and further al using
a heat-aler. Place bags in racks, and load carefully onto hydrocycler arm Note: Make sure bag is completely aled in order to prevent emulsion from spilling out into hydrocycler during PCR!
7. U the following conditions for PCR amplification:
Step Temperature
(ºC) Time
(min)
Cycles
1 95 7 1
2 95 1
3 60 2 35
4 72 1.5
5 72 12 1
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The annealing temperature (step 3) should be t to 2-5 ºC below the T m of the primer being ud
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Note:For cycling in a Peltier machine, u a cycle of: 95 ºC for 5’; 35 cycles of 95 ºC for 30s, 60 ºC for 60s, 72 ºC for 30s; 72 ºC for 10’; 4 ºC until the reactions are removed from the machine.体型最小的猫
8. Let reaction run till completion, or reaction can be left in hydrocycler O/N at 4ºC
9. Refer to 20mL emulsion breaking protocol for how to break emulsion
References:
This protocol is adapted from Williams et al. Amplification of complex gene libraries by emulsion PCR.Nature Methods 3(7):545-550 (2006) and from MYcroarray’s “Emulsion PCR for Oligonucleotide Library Amplification” protocol.
