Emulsion PCR

For initial amplification from Oligo-Library.

Setup

Components

• Glass vials kept at -20C.
• magnetic stir plates at 4C/in cold room (capable of 1000 rpm)
• emulsion oil mix (can make in 50mL volume and store at 4C):

1. 80 uL ABIL EM90 (request from cosmetic company)
2. 1 uL Triton-X 100
3. 1919 uL mineral oil

Total volume

100 uL aqueous PCR + 600 uL emulsion

4x Master mix (400 uL volume)

• 40 uL Buffer A
• 8 uL 10 mM dNTPs
• 4 uL undiluted LC library
• 20 uL 10 mg/mL BSA
• 4 uL KAPA Taq
• 321 uL dH2O

Procedure

setup emulsion

• vortex emuslion mix
• add 600 uL to tubes at 4C (use high viscosity pipettes)
• spin at 1000 RPM 1 min+
• add dropwise with a p200 pipette the aqueous PCR mix. Start timer 10 min.
• Remove from spinner after 10 min.
• Pipette emulsion mix into PCR tubes. Can use cut P200 tips with clean razor blade.

PCR:

1. 95C 1 min
2. 95C 15 seconds
3. 60C 15 seconds
4. 72C 20 seconds
5. rpt 2->4 29X
6. 72C 5 min
7. 4C forever.

emulsion breaking

1. Pool the emulsion PCR reactions in a 1.6 ml microcentrifuge tube.
2. Add 2µl of any kind of gel loading buffer to visualize the aqueous phase during recovery.
3. Add 200 µl of mineral oil and vortex 30 sec.
4. Centrifuge at 13,000g for 10 min.
5. Remove the oil (upper phase).
6. Add 1 ml of water-saturated diethyl ether and vortex 1 minute.
7. Centrifuge at 13,000g for 1 min.
8. Remove the diethyl ether (upper phase).
   1. Add 1 ml of water-saturated ethyl acetate and vortex 1 minute. Centrifuge at 13,000g for 1 min.
9. Remove the ethyl acetate (upper phase).
   1. Add 1 ml of water-saturated diethyl ether and vortex 1 minute. Centrifuge at 13,000g for 1 min.
10. Remove the diethyl ether (upper phase).
    1. Evaporate the residual diethyl ether by incubating the tube at 37 ˚C for 5 minutes with the cap open. An aliquot of the PCR product can be analyzed by electrophoresis at this step.

PCR cleanup

• use zippy PCR cleanup kit
• Add extra spin after last wash buffer (like Qiagen)
• elute in 12 uL ddH2O.

Proceed to PCR amplification for probe synthesis.