Hypothesis 2: Gene L is necessary for phage propagation.
- Unfortunately, both + and - PFU ligase samples showed amplification at ~> 5.3 kb dsDNA, so the ligase was not able to repair the nick to the amplified PHIX174 genome in parallel in the reaction.
- Next step is to make amplify a large batch of PHIX174 and PHIX174 T3485A.
- 2 × 50 μL WP-PCR reaction:
- 37 μL H2O
- 5 μL 10X reaction buffer
- 5 μL 100 μM primer 4 mix OR 20 μM primer 4 T3485A mix
- 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
- Cycling parameters:
- 95 °C 2 m
- 95 °C 30 s
- 58° C 30 s
- 72 °C 15 m
- Repeat 2-4 an additional 29 times for 30 total cycles
- 72 °C 30 m
Characterization B-C: Expression of PHIX174 promoters/UTRs fused to PX-UTR1-deGFP and PX-UTRX-deGFP.
- Spun down last night's midiculture in ~10 2 mL tubes (4 mL/tube) and stored at -40 °C for later use.
- Verification of pBEST-PX-MGapt plasmid set. I have 8 plasmids (6 promoters, 1 PL-PA tandem promoter, and 1 promoterless control). Verify by BamHI-XhoI double digest across the board.
- 100 μL BamHI-XhoI double digest @ 90%:
- 60 μL H2O
- 10 μL NEB Buffer 3
- 10 μL 10 mg/mL BSA
- 5 μL BamHI
- 5 μL XhoI
- Aliquot 8 × 9 μL
- 1 μL pBEST-PA-MGapt-T500
- 1 μL pBEST-PB-MGapt-T500
- 1 μL pBEST-PD-MGapt-T500
- 1 μL pBEST-PF-MGapt-T500
- 1 μL pBEST-PG-MGapt-T500
- 1 μL pBEST-PL-MGapt-T500
- 1 μL pBEST-PL-PA-MGapt-T500
- 1 μL pBEST-NONE-MGapt-T500
- Incubate 37 °C overnight.
|
PHIX174 Cell Free Expression
Main project page
Previous entry Next entry
