Team:BYU Provo/Notebook/SmallPhage/Springexp/Period2/Exp/5.20 Mutagen Concentration Experiment
From 2013.igem.org
(4 intermediate revisions not shown) | |||
Line 14: | Line 14: | ||
<font color="#333399" size="3" font face="Calibri"> | <font color="#333399" size="3" font face="Calibri"> | ||
- | : | + | <font size = "4"> |
+ | |||
+ | : <u> '''Small Phage''' </u> </font> | ||
: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]] | : [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]] | ||
Line 86: | Line 88: | ||
: We plated -4 dilution titers with x6 and x8 top agar using similar procedure to the previous selection test. | : We plated -4 dilution titers with x6 and x8 top agar using similar procedure to the previous selection test. | ||
- | 6) | + | 6) Selection - round 1 (6.7) |
+ | |||
+ | : From preliminary selection test - third round, we were able to determine that we need to plate at -4 dilution to have enough plaques per plate | ||
+ | |||
+ | : We plated 45 plates with -4 dilution of 200ug stock using x8 top agar. We also plated 5 plates with -3 dilution of 0ug stock using x8 top agar as control. | ||
+ | |||
+ | 7) Large plaque confirmation (6.10) | ||
+ | |||
+ | : From the 45 plates above we picked 12 of the biggest plaques and plated them using x8 top agar to confirm whether the supposed mutant have stable phenotype. Specifically, we added 50uL of LB to 12 eppendorf tubes. Each plaque was then stabbed with a pipet and the pipet was dipped into the 50uL LB; we pipetted up and down several times to ensure that phage particles got in the LB. | ||
+ | |||
+ | : To 12 test tubes, we added 0.75mL of Bl21 overnight and 30uL of phage in LB prepared in the previous step. They were incubated for 20 minutes. | ||
+ | |||
+ | : To each of the test tubes, 7mL of x8 top agar was added and then plated unto LB plates. | ||
+ | |||
+ | : The plates were incubated at 37C for approximately 24 hours. | ||
'''V) Results''' | '''V) Results''' | ||
Line 114: | Line 130: | ||
: -6 at x6 only showed a couple plaques on each plate. For -4 at x6, there were many plaques. It seems like the number of plaques increase as we increase the mutagen concentration. | : -6 at x6 only showed a couple plaques on each plate. For -4 at x6, there were many plaques. It seems like the number of plaques increase as we increase the mutagen concentration. | ||
- | + | [[File:MutagenPlate4.JPG|400px|center]] | |
+ | |||
+ | 5) Preliminary selection test - third round | ||
+ | |||
+ | : x6 top agar produced bigger/more plaques than x8 top agar. For selection purposes, we decided to use x8 top agar from now on. | ||
+ | |||
+ | 6) Selection - round 1 | ||
+ | |||
+ | : We saw a few bigger plaques (compared to the average plaque sizes) from the 45 plates. These are ones we are interested in. | ||
+ | |||
+ | [[File:MutagenPlate5.JPG|400px|center]] | ||
+ | |||
+ | 7) Large plaque confirmation | ||
+ | |||
+ | : Large scale contamination prevented any plaque from showing up. | ||
'''VI) Conclusion''' | '''VI) Conclusion''' | ||
- | + | Because there appears to be no visible decrease in the number of plaques as the concentration of mutagen increases, mutagenesis probably didn't occur effectively. In order to hopefully fix this, we will try mutagenesis again with a minimal media instead of LB. (See [[Team:BYU Provo/Notebook/SmallPhage/Springexp/Period3/Exp/6.12 Mutagen Concentration Test - Perfected Protocol|6.12 Mutagen Concentration Test - Perfected Protocol]]) | |
Latest revision as of 13:38, 9 September 2013
Small Phage May - June Notebook: Experiments
| ||
|
5.20 Mutagen Concentration Experiment
I) Purpose
II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Applying the mutagen (5.20)
2) Spot test (5.22)
3) Preliminary selection test (5.24)
4) Preliminary selection test - second round (5.29)
5) Preliminary selection test - third round (6.3)
6) Selection - round 1 (6.7)
7) Large plaque confirmation (6.10)
V) Results 1) Applying mutagen
2) Spot test
3) Preliminary selection test
4) Preliminary selection test 2
5) Preliminary selection test - third round
6) Selection - round 1
7) Large plaque confirmation
Because there appears to be no visible decrease in the number of plaques as the concentration of mutagen increases, mutagenesis probably didn't occur effectively. In order to hopefully fix this, we will try mutagenesis again with a minimal media instead of LB. (See 6.12 Mutagen Concentration Test - Perfected Protocol)
|