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Team:BYU Provo/Notebook/LargePhage/Springexp/Period2/Dailylog
From 2013.igem.org
| Large Phage May - June Notebook: May 13 - May 26 Daily Log
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5/13/13 Today we made stocks for our M9+ media. We made 1 L of 50X E-salts media that is stored in Dr. Grose’s lab. We also made a 100x stock of CaCl2 and NaCl which is stored in the iGEM classroom. To make M9+ media, mix: (makes 1 L) 20 mL 50X E-salts 10 mL 20% glucose (in Dr. Grose’s lab) 2.5 mL Ca/Na (iGEM room) 10g Casamino acids (need to order) Fill jar to 1 L with dH20 Autoclave
5/15/13 15 May 2013 KS BDM Today we got out mutagen 5’deoxy 2’deoxyuridine! We used the spectrophotometer to get OD readings on out bacteria to make sure that we got about 650 ul to 25 ml. We are going to use about 200 ug of mutagen for every 2 x 10^9 phage Today Bryan showed the large and small phage groups how to pour plates properly. Plaques/(dilution * amt infected with)
5/20/13 Today we need to run a dilution series to test the titer of our mutated phage stock. We also need to start selecting for small plaques and learning how to pick them and titer them out. We also should run a UV test on the mutated phage stock compared to the normal stock. We picked one plaque off of the 180 sec UV plate sample. We suspended it in 1 mL of broth, and then UV-ed 20 uL samples at 45 second intervals. The number of plaques decreased the longer the samples sat under UV light. The samples were irradiated from 0 sec to 4 min 30 sec. As a control, we diluted the T4Do stock to 10^-6 and tested 20 uL at 45 sec intervals (up to 6 min) under UV light. Also, we diluted the T4 mutagenized stock to 10^-6 and tested 20 uL at 45 sec intervals (up to 4 min 30 sec) under UV light. UV tests were done by placing 20 uL spots on parafilm and placed in a BSL-2 hood with the UV light turned on.
5/22/13 Results from 5/20/13       EVERY 1:30 Today we need to run a dilution series to test the titer of our mutated phage stock. We also need to start selecting for small plaques and learning how to pick them and titer them out. We also should run a UV test on the mutated phage stock compared to the normal stock. We picked one plaque off of the 180 sec UV plate sample. We suspended it in 1 mL of broth, and then UV-ed 20 uL samples at 45 second intervals. The number of plaques decreased the longer the samples sat under UV light. The samples were irradiated from 0 sec to 4 min 30 sec. As a control, we diluted the T4Do stock to 10^-6 and tested 20 uL at 45 sec intervals (up to 6 min) under UV light. Also, we diluted the T4 mutagenized stock to 10^-6 and tested 20 uL at 45 sec intervals (up to 4 min 30 sec) under UV light. UV tests were done by placing 20 uL spots on parafilm and placed in a BSL-2 hood with the UV light turned on.
5/24/13 Results from 5/22/13 - We left the plates in the incubator for 48 hours, which caused contamination on many plates to grow. When checked at 24 hours, the T4 mutagenized stock had a web plate at 10^-3 and less than 5 plaques at 10^-6. This experiment will need to be redone from 10^0 down through 10^-6. The whole mutagenesis may need to be redone if this only represents a dilution of our titer when we were trying to grow it in liquid culture. (We infected with ___ mL of phage at ___ titer in ____ vol of resuspended bacteria.) Under UV light, the T4Do stock (diluted to 10^-6) has 19 plaques after being irradiated for six minutes (down from almost cleared at 0 min). Under UV light, the 180 sec UV plate spot (diluted in 1 mL) has a few hundred plaques on it, but the amount dropped significantly at 4 min 30 sec from when it was UV-ed for 0 min. We will re-titer the T4-Mut stock so we can learn whether it was diluted or whether an infection worked. We will also re-run the UV test comparing the T4-Mut (10^-3) with the T4-Do stock (at 10^-6) for survivability. The mutagenized phage should survive better. Since the loss of plaques seemed to level off for the T4Do stock at 5:15 (23 plaques) and 6 min (19 plaques), we will test a 7 min and 8 min timepoint to see if it stays level, suggesting these phage have multiple genomes and are severely mutated.             
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