(b) Step 2: If screening multiple colonies, prepare a reaction
master mix by multiplying by the number of colonies to
be screened plus negative and positive controls (Example:
19 + 1 + 1 ¼ 21 + 10% volume pipetting error).
(c) Step 3: Spin down and aliquot the master mix in a 0.2 mL
microcentrifuge tube.
(d) Step 4: Perform the PCR reaction as follows, denature the
template at 95
C for 3 min, followed by 35 cycles of
95
C for 30 s, 55
C for 30 s and 72
C for 2 min. Then
incubate at 72
C for 5 min to complete the reaction.
Negative control (no template colony) and positive control (an aliquot of the original purified PCR product)
should be run in parallel.
11. Examine the PCR products by gel electrophoresis with 1%
agarose in 1Â TAE buffer.
12. Select a positive colony containing the recombinant plasmid
and inoculate in 5 mL LB broth containing 100 μg/mL ampicillin. Then incubate at 37
C with constant agitation at
220 rpm for 16 h.
13. Extract the plasmid using FavorPrep™ Plasmid Extraction
Mini Kit by following the standard manufacturer protocol
and measure the concentration using a NanoDrop
spectrophotometer.
14. Dilute the plasmid to 100 ng/μL using nuclease-free water.
15. Perform restriction enzyme digestion to determine the correct
clone as follows.
Fig. 1 Colony PCR screening. An ordered array of putative transformants. Each
grid square contains one randomly selected transformant that will be screened
by colony PCR
Zika Virus-Like Particles
195
master mix by multiplying by the number of colonies to
be screened plus negative and positive controls (Example:
19 + 1 + 1 ¼ 21 + 10% volume pipetting error).
(c) Step 3: Spin down and aliquot the master mix in a 0.2 mL
microcentrifuge tube.
(d) Step 4: Perform the PCR reaction as follows, denature the
template at 95
C for 3 min, followed by 35 cycles of
95
C for 30 s, 55
C for 30 s and 72
C for 2 min. Then
incubate at 72
C for 5 min to complete the reaction.
Negative control (no template colony) and positive control (an aliquot of the original purified PCR product)
should be run in parallel.
11. Examine the PCR products by gel electrophoresis with 1%
agarose in 1Â TAE buffer.
12. Select a positive colony containing the recombinant plasmid
and inoculate in 5 mL LB broth containing 100 μg/mL ampicillin. Then incubate at 37
C with constant agitation at
220 rpm for 16 h.
13. Extract the plasmid using FavorPrep™ Plasmid Extraction
Mini Kit by following the standard manufacturer protocol
and measure the concentration using a NanoDrop
spectrophotometer.
14. Dilute the plasmid to 100 ng/μL using nuclease-free water.
15. Perform restriction enzyme digestion to determine the correct
clone as follows.
Fig. 1 Colony PCR screening. An ordered array of putative transformants. Each
grid square contains one randomly selected transformant that will be screened
by colony PCR
Zika Virus-Like Particles
195