Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.14 Mutagen Concentration Test - Seventh Protocol
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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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* Variation exist on every plate. Generally, aliquots with smaller number showed the smaller plaques correlating with bigger phage that migrated to the bottom of the gradient. In contrast, aliquots with bigger number showed bigger plaques correlation with smaller phage that didn't move too far down the gradient. 12 small plaques and 12 big plaques are the respective end of the spectrum were selected (see picture below for more graphic description). Each plaque was picked with a pipet tip and dipped into 50uL of LB. | * Variation exist on every plate. Generally, aliquots with smaller number showed the smaller plaques correlating with bigger phage that migrated to the bottom of the gradient. In contrast, aliquots with bigger number showed bigger plaques correlation with smaller phage that didn't move too far down the gradient. 12 small plaques and 12 big plaques are the respective end of the spectrum were selected (see picture below for more graphic description). Each plaque was picked with a pipet tip and dipped into 50uL of LB. | ||
- | * The picked plaques were plated. Specifically, 0.5mL of E coli B was infected with 20uL of collected plaque sample (in LB) for approximately 15 minutes before they were plated along with 6 mL of x6 top agar. | + | * The picked plaques were plated. Specifically, 0.5mL of E coli B was infected with 20uL of collected plaque sample (in LB) for approximately 15 minutes before they were plated along with 6 mL of x6 top agar. This step was repeated after no bacteria or phage plaques showed up the first time. |
'''V) Results''' | '''V) Results''' | ||
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[[File:BYUSPM7-4.JPG|350px|link=]] | [[File:BYUSPM7-4.JPG|350px|link=]] | ||
[[File:BYUSPM7-5.JPG|350px|link=]] | [[File:BYUSPM7-5.JPG|350px|link=]] | ||
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+ | * Plating of selected plaques | ||
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+ | : This step was repeated twice. On both tries, no plaque or bacteria showed up for the samples. However, wild type plating showed normal/expected results. Most likely, picking plaques with pipet tips and dipping it into LB created stock too concentrated that all of the bacteria was lysed during incubation. | ||
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+ | [[File:BYUSPM7-6.JPG|350px|center|link=]] | ||
'''VI) Conclusion''' | '''VI) Conclusion''' | ||
+ | * The CsCl gradient in this round of mutagenesis is not optimal for two reasons: | ||
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+ | : 1) Contamination between layers was obvious during the collection process. | ||
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+ | : 2) No definitive way of relating aliquot to each gradient due to randomly collecting certain volume of phage CsCl suspension. | ||
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+ | * Despite these setbacks we still saw a certain variance in phage plaque sizes. Although the characterization process, in which we meant to verify whether or not the phenotype is stable, did not work so well, we are optimistic about the direction we are going. We have already started a new round of mutagenesis: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp/8.26 Mutagen Concentration Test|8.26 Mutagen Concentration Test - Eighth Protocol]]. This time, we will perfect every step that we had trouble with this time. | ||
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Latest revision as of 15:39, 9 September 2013
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8.14 Mutagen Concentration Test - Seventh Protocol
I) Purpose To mutate T7 phage for different capsid sizes. II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Overnight (The day before) (8.13)
2) Applying the mutagen (8.14)
3) Spot test to determine phage concentration (8.14)
4) CsCl gradient (8.15)
5) Characterizing post-CsCl phage (8.16-8.18)
V) Results 2) Applying the mutagen
3) Spot test to determine phage concentration
5) Characterizing post-CsCl phage
VI) Conclusion
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