Hypothesis 2: Gene L is necessary for phage propagation.
- Gel electrophoresis showed the following over the rest of the primer 4 mix range:
- Nothing the the column of -primer control.
- A strong band at > 5kb at 0.5 μM primer (band 1), growing weaker as primer concentration increased, and disappearing for the 4 and 8 μM primer columns. Band 1 = amplified, nicked ΦX174 genome.
- A weak band at < 100 bp (band 2) at 0.5 μM primer (band 2), growing stronger as primer concentration increased. Band 2 = ssDNA primers.
- A weak band at < 100 bp (band 3) > band 2, not apparent until 1 μM (nothing at 0.5 μM) and growing stronger with increasing primer concentration. Band 3: hybridized ssDNA primers.
- Therefore, as I anticipated, too much primers inhibited PCR due to primer hybridization of complementary primers used for WP-PCR.
- 0.5 μM should be used for WP-PCR amplification of ΦX174 genome. I finalized the WP-PCR protocol. Other long WP-PCRs should use between 0.1 and 1 μM primer, with the optimal primer concentration being found for each new primer/template combination.
- Final WP-PCR protocol is ...
- 50 μL WP-PCR reaction:
- 37 μL H2O
- 5 μL 10X reaction buffer
- 5 μL 5 μM primer mix (0.5 μM final, each)
- 1.5 μL 3.2 nM ΦX174 template (~ 0.1 nM final)
- 0.5 μL 25 mM dNTPs mix
- 1 μL PfuUltra I DNA polymerase
Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.
- Re-concentrated pBEST-Pr-MG-apt-UTR1-deGFP-T500. Quantifluore measured concentration to be 231.5 nM (1.6%).
- Repeat cell-free expression, now with top plasmid concentration point actually at 10. Also, do it at gain range of 200, 225, and 250.
- Cell-free expression 80%:
- 30 μL extract (12May11)
- 1.17 μL water
- 0.9 μL 100 mM Mg-glutamate (1 mM final)
- 2 μL 3 M K-glutamate (66.7 mM final)
- 22.5 μL 6 mM amino acids mix (1.5 mM final)
- 6.43 μL 14X 3-PGC buffer (1X final)
- 4.5 μL PEG8000 40% (2% final)
- 4.5 μL 200 μM malachite green (10 μM final)
- Aliquot 6 × 8 μL into 394-well plate and then add plasmid (pBEST-Pr-MGapt-UTR1-deGFP-T500) range:
- 2 μL water = 0 nM
- 2 μL 50 nM / 3^4 * 20% = .123 nM
- 2 μL 50 nM / 3^3 * 20% = .370 nM
- 2 μL 50 nM / 3^2 * 20% = 1.11 nM
- 2 μL 50 nM / 3^1 * 20% = 3.33 nM
- 2 μL 50 nM / 3^0 * 20% = 10 nM
- Plate reader:
- 29 °C
- Shake: fast double orbital 30s
- Read: 625/655 nM @ gain = 200 w/ settings
- Read: 625/655 nM @ gain = 225 w/ settings
- Read: 625/655 nM @ gain = 250 w/ settings
- Loop: 2-5 every 3 m for 12 h
- settings = full plate, high lamp energy, endpoint, bottom reading, other settings default
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PHIX174 Cell Free Expression
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