Team:BYU Provo/Notebook/SmallPhage/Springexp/Period3/Exp/6.12 Mutagen Concentration Test - Perfected Protocol
From 2013.igem.org
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+ | : <u> '''Small Phage''' </u> </font> | ||
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]] | : [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]] | ||
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- | <font size="5"> '''6.12 Mutagen Concentration Test - | + | <font size="5"> '''6.12 Mutagen Concentration Test - Second Protocol''' </font> |
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: - For each dilution, a test tube was assigned to it. To each test tube, 0.75mL of BL21 liquid culture overnight was added and then transfected with 30uL of phage dilution. After 20 minutes of incubation, 7mL of x8 agar was added to each test tube and the content was then plated | : - For each dilution, a test tube was assigned to it. To each test tube, 0.75mL of BL21 liquid culture overnight was added and then transfected with 30uL of phage dilution. After 20 minutes of incubation, 7mL of x8 agar was added to each test tube and the content was then plated | ||
- | 3) Selection | + | 3) Selection Test 1 (6.17) |
: - Titering result revealed an adequate plaque number at -2 dilution for each mutagen concentration. We thus plate 50 plates using 500ug -2 dilution and x8 agar. As control, 5 plates were plated using 0ug -2 dilution and x8 agar. | : - Titering result revealed an adequate plaque number at -2 dilution for each mutagen concentration. We thus plate 50 plates using 500ug -2 dilution and x8 agar. As control, 5 plates were plated using 0ug -2 dilution and x8 agar. | ||
- | 4) | + | 4) Large Plaque Confirmation 1 (6.19) |
- | : | + | : - We selected 12 of the largest plaques that we could find from our Selection Test 1 |
+ | : - For each plaque, we scrapped off of the plaque with a pipet tip and then dipped it in 100ul of LB in an eppendorf tube. | ||
+ | : - To 12 test tubes each, we added 750ul of BL21 overnight. | ||
+ | : - We then added 30ul of phage from each eppendorf tube into one of the 12 test tubes and let it incubate at room temperature for 20 minutes. | ||
+ | : - Next, we added 7ml of x8 top agar to each test tube and plated it on 12 LB plates. | ||
+ | : - Placed in 37 C incubator for about 24 hours. | ||
- | + | 5) Large Plaque Confirmation 2 (6.24) | |
- | + | : - Using the 70uL (100uL - the 30uL used) stock from 6.19, we performed dilution series 1:100 to generate -2 and -4 stock. | |
+ | : - 0, -2, and -4 stock from each plaque was plated using 0.75mL of BL21 overnight, 30uL of phage, and 7mL of x8 top agar. Similar procedures as those in 6.19 were used. | ||
+ | : - The plates were incubated in 37 C incubator for approximately 24 hours. | ||
- | + | '''V) Results''' | |
- | + | 1) Applying the mutagen | |
- | + | : No obvious signs of clearage was seen after 30 minutes of incubation with phage. | |
+ | 2) Titer to determine phage concentration | ||
+ | |||
+ | : There was a little variation in the number of plaques as the mutagen concentration increased, but there was no clear sign of phage death due to the mutagen. Each dilution showed plaques up to -3. | ||
+ | |||
+ | [[File:Mutagen2Plate1.JPG|400px|center]] | ||
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+ | 3) Selection Test 1 | ||
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+ | : Each plate had roughly 25 to 50 plaques on it with natural variation in plaque sizes. From all these plaques, we selected 12 that appeared to be bigger than normal. | ||
+ | |||
+ | [[File:Mutagen2Plate2.JPG|400px|center]] | ||
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+ | 4) Large Plaque Confirmation 1 | ||
+ | |||
+ | : All the plates seemed to have been completely or almost cleared by the phage, leaving no visible plaque sizes. Also, the plates were contaminated. | ||
+ | |||
+ | [[File:Mutagen2Plate3.JPG|400px|center]] | ||
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+ | 5) Large Plaque Confirmation 2 | ||
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+ | : No abnormally large plaque detected. Plaques are actually smaller than the ones we originally picked from. This could most likely be due to differences in agar concentration. | ||
+ | |||
+ | [[File:Mutagen2Plate4.JPG|400px|center]] | ||
+ | [[File:Mutagen2Plate5.JPG|400px|center]] | ||
'''VI) Conclusion''' | '''VI) Conclusion''' | ||
+ | |||
+ | We decided that mutagenesis did not occur properly again because there was no evidence of phage death with increasing mutagen concentration and the large plaques that we isolated did not retain their size once they were replicated. Poor mutagenesis probably occurred because we only incubated the phage for 30 minutes. In order to attempt to fix this, we will redo mutagenesis, but this time we will incubate the phage until clearage. | ||
Latest revision as of 15:30, 9 September 2013
Small Phage May - June Notebook: Experiments
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6.12 Mutagen Concentration Test - Second Protocol
I) Purpose
II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Applying the mutagen (6.12)
2) Titer to determine phage concentration (6.14)
3) Selection Test 1 (6.17)
4) Large Plaque Confirmation 1 (6.19)
5) Large Plaque Confirmation 2 (6.24)
V) Results 1) Applying the mutagen
2) Titer to determine phage concentration
3) Selection Test 1
4) Large Plaque Confirmation 1
5) Large Plaque Confirmation 2
VI) Conclusion We decided that mutagenesis did not occur properly again because there was no evidence of phage death with increasing mutagen concentration and the large plaques that we isolated did not retain their size once they were replicated. Poor mutagenesis probably occurred because we only incubated the phage for 30 minutes. In order to attempt to fix this, we will redo mutagenesis, but this time we will incubate the phage until clearage.
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