User:Sean P Corum/Notebook/PHIX174 Cell Free/2012/06/19

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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:
  1. pBEST-BamHI//XhoI-T500 dsDNA: ~≥ 2500b (expected 2464)
  2. PCR product: ~≤ 1000 bp (expected 861)
  3. Cleaned PCR product: ~≤ 1000 bp = same as 2 (expected 861)
  4. 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:
  1. 34.5 μL ddH2O
  2. 5 μL 10X reaction buffer
  3. 2 μL (10 ng) pWhitscript 4.5kb control plasmid template
  4. 1.25 μL (125 ng) control primer 1 (34b)
  5. 1.25 μL (125 ng) control primer 2 (34b)
  6. 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:

  1. 95 °C 60 s
  2. 95 °C 30 s
  3. 55 °C 30 s
  4. 68 °C 10 min
  5. Repeat 2-4 a total of 20 times.
  6. 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

  • None.

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