 PHIX174 Cell Free Expression
|
 Main project page
 Previous entry Next entry
|
Cell Free Expression of PHIX174: June 19, 2012
- SC 21:27, 19 June 2012 (EDT):
Characterization B: Expression of PHIX174 promoters fused to UTR1-deGFP.
- I continued with the construction of pBEST-PA-UTR1-deGFP-T500 by performing PCR Purification of yesterday's PCR product, followed by volume reduction to 17 μL via vacuum centrifuge.
- I then digested the PCR product with BamHI and XhoI at 37 °C for 3 hr.
- Finally, I purified the digestion product from a gel to obtain the BamHI-UTR1-deGFP-XhoI linker, followed by volume reduction by vacuum centrifuge. (I accidentally fully dried the sample and then re-hydrated it. We'll see if this works out at all.)
- Gel electrophoresis of all these steps as well as the vector backbone indicated the presence of the following dsDNAs:
- pBEST-BamHI//XhoI-T500 dsDNA: ~≥ 2500b (expected 2464)
- PCR product: ~≤ 1000 bp (expected 861)
- Cleaned PCR product: ~≤ 1000 bp = same as 2 (expected 861)
- Digestion product (BamHI-UTR1-deGFP-XhoI linker): brighter band ~< length of both 2&3, very dim band << 500 bp (expected 725 bp, 130 bp, 6 bp)
- Since all dsDNAs are the correct size, I went ahead and performed ligation to form pBEST-PA-UTR1-deGFP-T500.
- Tomorrow, I will proceed with transformation into JM109 competent cells.
Hypothesis 2: Gene L is necessary for phage propagation.
Since I haven't been able to amplify PHIX174 via whole plasmid PCR, I investigated the following options:
- Perform whole plasmid PCR using the controls included in the QuickchangeII Kit (Agilent). According to the manual, a 50 μL control reaction includes the following components:
- 34.5 μL ddH2O
- 5 μL 10X reaction buffer
- 2 μL (10 ng) pWhitscript 4.5kb control plasmid template
- 1.25 μL (125 ng) control primer 1 (34b)
- 1.25 μL (125 ng) control primer 2 (34b)
- 5 μL dNTP mix (2mM each)
I combined these components and then divided in half. In half I put 0.5 μL PFU Ultra HF DNAP, and in the other I 0.5 μL ddH2O (control). I then ran the following thermocycling program:
- 95 °C 60 s
- 95 °C 30 s
- 55 °C 30 s
- 68 °C 10 min
- Repeat 2-4 a total of 20 times.
- 12 °C hold
- Research alternative whole plasmid PCR protocols. On hold until results of Quickchange II control experiment are in. Here is one possible lead.
- Investigate possible E. coli amplification of PHIX174. On hold until results of Quickchange II control experiment are in.
|
Notes
Recently Edited Notebook Pages
PHIX174 Cell Free Expression
