Team:BYU Provo/Notebook/LargePhage/Summerexp/Period10/Dailylog


Large Phage September - October Notebook: October 1 - October 15 Daily Log

Large Phage










Today we planned out a schedule that we will need to follow closely in order to have more data to present at the world jamboree at MIT. Our goals are to: Isolate a new large phage by running multiple new mutagenesis procedures Characterize mutations using PCR Clone the MCP and Capsid Vertex Protein into the registry (which requires point mutations to remove restriction sites)




Today we designed primers for the mutagenesis we'll need to do to remove illegal restriction sites in the T4 MCP and capsid vertex proteins.

We also ran PCR on plaques from T4 WT and from liquid from sample 21-7 which was our small mutant that was supposed to be large. The PCR was run using these ratios:

To a eppendorf tube, add 120 ul ddH20 15 ul 10X TAQ Buffer 4.5 ul 10mM dNTP's 3 ul of forward primer 3 ul of reverse primer 1.5 uL Taq Polymerase

50 uL in each PCR tube

_ uL of template


Today we need to:
- Run a gel of our PCR products
We'll use a 0.4% gel... next time we can use 1% instead.
- Start culturing our picked plaque (T4 WT to be used for mutagenesis)
We added 1 mL of E. coli B to 25 mL of broth and let it incubate. When it was more turbid we added 0.5 mL of broth that we had picked a plaque and diluted it in. We let this incubate for ___ hours.

10/15/13 Description - Flask - 9am - 11am - 4pm

T4 Wild type stock - Flask 1 - 25 mL LB Broth, 1 loop E. coli - 1 mL T4 phage

1x mutagen in LB - Flask 2 - 25 mL of LB broth, 100 uL adenine, 1 loop E. coli - 100 uL adenine, 200 uL uracil, 500 uL BrdU, 1 mL T4 phage

1x mutagen in M9 - Flask 3 - 25 mL of M9+ broth, 100 uL adenine, 1 loop E. coli - 100 uL adenine, 200 uL uracil, 500 uL BrdU, 1 mL T4 phage

2x mutagen in M9 - Flask 4 - 25 mL of M9+ broth, 100 uL adenine, 1 loop E. coli - 100 uL adenine, 200 uL uracil, 1 mL BrdU, 1 mL T4 phage

We inoculated a 250 uL flask with 25 mL of LB broth, 100 uL (20 ug/mL) of adenine, and a loop of E. coli. We inoculated 25 mL of M9 with the same ingredients. We let these flasks incubate for ~2 hours until moderately turbid. After the bacteria is moderately turbid, we added another 100 uL of adenine and 200ul (20 ug/mL) uracil to the 25ml flasks of E. coli B in LB and in M9 along with 500 ul (200 ug/mL) mutagen 5-bromo 2-deoxyuridine and 500 ul T4 phage (1x10^9 pfu/mL) and put that in the shaker to determine if the flask will clear in about three hours. After 3 hours, we removed 5 mL of bacterial culture and placed it in a 15 mL tube with 1 mL of chloroform. The LB culture cleared very quickly, while the M9 culture did not clear very well. We should wait for the M9 to work. We pelleted the bacteria in a centrifuge at 3000 rpm for 10 minutes and resuspended each pellet in 2 mL of broth. We transferred it to a 15 mL tube and added 10 uL of nuclease mix, 5 uL of 1 M MgCl2, and 2 mL of chloroform. We mixed it gently and let it incubate for 30 minutes. Then, we centrifuged it at 2700 rpm for 10 minutes. The supernatant was removed and transferred to a new 15 mL tube.