Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period11/Dailylog

From 2013.igem.org

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{| width="100%"
{| width="100%"
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| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Small Phage May - June Notebook: May 1 - May 12 Daily Log'''</font>
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| colspan="3" | <font color="#333399" size="5" font face="Calibri">  
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: '''Phage Purification July - August Notebook: August 17 - August 31 Daily Log'''</font>
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<font color="#333399" size="3" font face="Calibri">
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: [[Team:BYU_Provo/Small_Phage|Overview]]
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<font size = "4">
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: [[Team:BYU Provo/Notebook/SmallPhage/Winterexp|March-April]]
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: <u> '''Phage Purification''' </u> </font>
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: [[Team:BYU Provo/Notebook/SmallPhage/Springexp|May-June]]
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: [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Summerexp|July-August]]
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: [[Team:BYU Provo/Notebook/Phage_Purification/Springexp|May-June]]
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: [[Team:BYU Provo/Notebook/SmallPhage/Fallexp|September-October]]
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: [[Team:BYU Provo/Notebook/Phage_Purification/Summerexp|July-August]]
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: [[Team:BYU Provo/Notebook/Phage_Purification/Fallexp|September-October]]
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<font size="4"> '''5/1/13''' </font>
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- Commencement of spring!!!
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<font face="Calibri" size="3">
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- Discussed goals and outlined plans for spring term
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<br>
<br>
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<html> <a href="https://2013.igem.org/Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period10/Dailylog"><< Previous</a>
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<font size="4"> '''5/2/13''' </font>
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<a href="https://2013.igem.org/Team:BYU_Provo/Notebook/Phage_Purification/Fallexp/Period1/Dailylog" style="display:block;float:right;">Next >></a>
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- Designed primers for amplifying and sequencing phage capsid protein
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</html>
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- Wrote emails inquiring mutagen (NG) and plasmid for in vitro assembly
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<br>
<br>
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<font size="4"> '''5/3/13''' </font>
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<font size="4"> '''8/19/13''' </font>
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- Performed agar test, focusing primarily on ×8
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- Performed a cesium chloride gradient on T4.  The gradient did not show any band and we aren't sure where the phage are in the gradient.  It is possible that we mixed the gradient before or after centrifugation which could have destroyed the band.
 +
:[[Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period1/Exp/8.19CsClGradient| 8.19 CsCl gradient]]
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: [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU_Provo/5.3 T7 phage selection method test|5.3 T7 phage selection method test]]
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AB DL AC
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- Processed phage amplification
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: [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU_Provo/5.3 Phage Amplification/Purification|5.3 T7 phage amplification/purification]]
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<br>
<br>
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<font size="4"> '''5/4/13''' </font>
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<font size="4"> '''8/21/13''' </font>
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- Performed dilution series using stock 5.3 (-1 through -11)
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- Discussed and weighed out the T4 cesium chloride gradient for next class. 
 +
We also discussed a new gradient for T7. We are hoping this gradient will narrow down exactly where the mutant phage will band and where the wild type phage will band.  We feel that the phage will differentiate around the 1.3 concentration.  By creating a gradient with small steps both above and below 1.3 we should be able to separate the phage out.
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- Started two 5mL overnights of BL21
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AB DL AC
<br>
<br>
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<font size="4"> '''5/5/13''' </font>
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<font size="4"> '''8/23/13''' </font>
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- Spot test using stock 5.3 and its dilution series (from 5.4)
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- Prepared a Cesium Chloride Gradient for T4
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:[[Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period1/Exp/8.23CsClGradient| 8.23 CsCl gradient]]
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: [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU_Provo/5.3 Phage Amplification/Purification|5.3 T7 phage amplification/purification]]
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AB DL AC
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- Started liquid culture for purification team (at around noon)
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: 1mL of BL21 overnight + 4mL of LB + 100μL -2 phage (received stock)
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- Started 5.5 amplification from a plaque test
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: [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU_Provo/5.5_Amplification_from_a_plaque_test|5.5 Amplification from a plaque test]]
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<br>
<br>
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<font size="4"> '''5/6/13''' </font>
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<font size="4"> '''8/26/13''' </font>
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- Continued [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU_Provo/5.3 T7 phage selection method test|5.3 T7 phage amplification/purification]]
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- Reviewed results for the T4 gradient. 
 +
-Ran CsCl Gradient on T7.
 +
:[[Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period1/Exp/8.26 CsCl Gradient| 8.26 T7 CsCl gradient]]
 +
We are having issues getting the T7 to band.  When we remove the gradient after centrifuging we are unable to see any band at all.  A spot test reveals phage are found all over the gradient.  We have had this same result several times and believe that the cesium chloride we are using could be the factor.  Our plan is to do a very broad test and see if we can get the phage to band anywhere.
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- Performed liquid culture phage concentration test
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AB DL AC
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: [[Team:BYU Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU Provo/5.6 T7+ Liquid Culture Phage Concentration Test|5.6 T7+ Liquid Culture Phage Concentration Test]]
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<br>
<br>
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<font size="4"> '''5/7/13''' </font>
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<font size="4"> '''8/28/13''' </font>
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- Started two 5mL of E coli BL21 overnight
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- Discussed the possible gradients we could perform to get T7 to band.  We are thinking of running a wild type gradient with different concentrations at 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and 1.7.  This should give us a rough estimate of where the wild type will band.
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- Designed procedure for applying mutagen and selecting for T7
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AB DL AC
<br>
<br>
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<font size="4"> '''5/8/13''' </font>
 
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- Went over procedure for applying mutagen and PCR with Dr. Grose
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</font>
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- Performed spot tests under [[Team:BYU Provo/Notebook/SmallPhage/Springexp/Period1/Exp/Team:BYU Provo/5.6 T7+ Liquid Culture Phage Concentration Test|5.6 T7+ Liquid Culture Phage Concentration Test]]
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<html> <a href="https://2013.igem.org/Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period10/Dailylog"><< Previous</a>
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- Started two 5mL BL21 overnights
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<a href="https://2013.igem.org/Team:BYU_Provo/Notebook/Phage_Purification/Fallexp/Period1/Dailylog" style="display:block;float:right;">Next >></a>
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</html>
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- Learned how to create LB plates
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<br>
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<font size="4"> '''5/9/13''' </font>
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- Practiced with ×6 and ×8 top agar
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: [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period1/Exp/5.9_T7_selection_method_test|5.9 T7 Selection Method Test #2]]
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-
 
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- Started [[Team:BYU Provo/Notebook/SmallPhage/Springexp/Period1/Exp/5.9 T7+ Liquid Culture Phage Concentration Test|5.9 T7+ Liquid Culture Phage Concentration Test #2]]
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- Discussed plans and schedule for next week.
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<br>
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<font size="4"> '''5/10/13''' </font>
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- Worked on [[Team:BYU_Provo/Notebook/SmallPhage/Springexp/Period1/PR|Progress Report]]
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<br>
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<font size="4"> '''5/12/13''' </font>
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- Started two 5mL overnight at around 6pm
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<br>
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</font>
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{{TeamBYUProvoFooter}}
{{TeamBYUProvoFooter}}

Latest revision as of 00:43, 28 September 2013


Phage Purification July - August Notebook: August 17 - August 31 Daily Log



Phage Purification
March-April
May-June
July-August
September-October



<< Previous Next >>


8/19/13

- Performed a cesium chloride gradient on T4. The gradient did not show any band and we aren't sure where the phage are in the gradient. It is possible that we mixed the gradient before or after centrifugation which could have destroyed the band.

8.19 CsCl gradient

AB DL AC


8/21/13

- Discussed and weighed out the T4 cesium chloride gradient for next class. We also discussed a new gradient for T7. We are hoping this gradient will narrow down exactly where the mutant phage will band and where the wild type phage will band. We feel that the phage will differentiate around the 1.3 concentration. By creating a gradient with small steps both above and below 1.3 we should be able to separate the phage out.

AB DL AC


8/23/13

- Prepared a Cesium Chloride Gradient for T4

8.23 CsCl gradient

AB DL AC


8/26/13

- Reviewed results for the T4 gradient. -Ran CsCl Gradient on T7.

8.26 T7 CsCl gradient

We are having issues getting the T7 to band. When we remove the gradient after centrifuging we are unable to see any band at all. A spot test reveals phage are found all over the gradient. We have had this same result several times and believe that the cesium chloride we are using could be the factor. Our plan is to do a very broad test and see if we can get the phage to band anywhere.

AB DL AC


8/28/13

- Discussed the possible gradients we could perform to get T7 to band. We are thinking of running a wild type gradient with different concentrations at 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, and 1.7. This should give us a rough estimate of where the wild type will band.

AB DL AC



<< Previous Next >>

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