Team:BYU Provo/Notebook/Phage Purification/Springexp/Period2/PR

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Revision as of 00:23, 4 June 2013

Small Phage May - June Notebook: May 13 - May 26 Progress Report

1. Goals for the week

Our goals for this week are to correctly prepare and successfully extract phage from a CsCl gradient.

2.Experiments and results

After our first attempt at purification using PEG, we decided to perform a phage titer to check for phage viability. The titers were successful. Both plates showed plaques. On the plate of W3110 infected with T4 phage, there was contamination. Plaques were still visible and it was determined that the team should continue on, as the next step of a CsCl gradient should remove any contaminants.

When performing the next step of the CsCl gradient, we added the incorrect amount of CsCl to phage suspension buffer, causing the gradient to be half as dense as it should have been. We had layered in very little phage to begin with, and the team determined it would be better to restart with a larger amount of phage particles to purify.

We completed the first step in PEG purification for the second time. With the doubled amount of phage, we had much more to layer in the CsCl gradient and there were visible bands in the gradient since the correct concentrations were used.

The CsCl gradient was successful the second time it was ran. There were visible bands of bacterial debris and phage particles.

3. Next Steps

Successfully extract phage particles from the CsCl gradient, remove from CsCl using phage suspension buffer.

Perform the third and final step of purification with an equilibrium CsCl gradient.

BeginPreparing for the preperation of Ghost Particles.

@@ Line 32: / Line 32: @@
 '''1. Goals for the week '''
-: Our two main goals for this week were to determine the mutagen concentration to use during mutagenesis, and to amplify the T7 minor capsid protein with PCR.
+: Our goals for this week are to correctly prepare and successfully extract phage from a CsCl gradient.
 '''2.Experiments and results '''
-.9 T7+ Liquid Culture Phage Concentration Test #2
+:After our first attempt at purification using PEG, we decided to perform a phage titer to check for phage viability. The titers were successful. Both plates showed plaques. On the plate of W3110 infected with T4 phage, there was contamination. Plaques were still visible and it was determined that the team should continue on, as the next step of a CsCl gradient should remove any contaminants.
-: We infected E. coli BL21 with different concentrations (1ul, 10ul, and 100ul) of phage to see which would yield the highest titer.  Plaques formed up to -8 on all the spot tests, showing that the concentration difference had no effect on the titer.  However, the plates were badly contaminated.
+:When performing the next step of the CsCl gradient, we added the incorrect amount of CsCl to phage suspension buffer, causing the gradient to be half as dense as it should have been. We had layered in very little phage to begin with, and the team determined it would be better to restart with a larger amount of phage particles to purify.
+:We completed the first step in PEG purification for the second time. With the doubled amount of phage, we had much more to layer in the CsCl gradient and there were visible bands in the gradient since the correct concentrations were used.
+:The CsCl gradient was successful the second time it was ran. There were visible bands of bacterial debris and phage particles.
-.13 Determining E. coli Concentration With Spectrophotometer
+'''3. Next Steps'''
-: To check if we could determine E. coli concentration with a spectrophotometer, we created a 7/5 dilution series of E. coli.  We then measured the absorption at 600nm of each E. coli dilution.  When plotted, these measurements showed a linear relationship between E. coli concentration and the absorption reading.
+:Successfully extract phage particles from the CsCl gradient, remove from CsCl using phage suspension buffer.
-: [[File:Spec.png]]
+: Perform the third and final step of purification with an equilibrium CsCl gradient.
-.15 Titer Test on 5.3 T7 new Phage Stock
-: Created a 1:10 dilution series with 5.3 T7 stock.  We then created titers of -5, -6, -7, and -8.  -5, -6, and -7 plates all had overlapping plaques.  However, the -8 plate had 7 plaques, giving us an estimated phage concentration of 7E8 particles/20ul.
-.20 Mutagen Concentration Experiment
-: In order to determine the best concentration of mutagen to use, we infected the E. coli in 5 tubes with 0ul, 10ul, 50ul, 100ul, and 200ul of our mutagen, 5-bromodeoxyuridine.  We then added 6uL of 5.3 phage stock to each tube, allowed it to incubate for 20 minutes, and purified the phage.  Next, we made a dilution series and performed a spot test and found that when the mutagen concentration is increased, the concentration of phage is decreased.
-[[File:MutagenPlate1.JPG|400px|center]]
-We then performed titers from -6, -7, and -8 of phage from our dilution series with x8 top agar.  There are a couple plaques on each plate.
-[[File:MutagenPlate3.JPG|400px|center]]
-.20 T7 Minor Capsid Protein PCR
-: In order to sequence the T7 minor capsid protein, we had to amplify the gene using PCR. We first isolated the DNA by boiling phage in a PCR machine and centrifuging it for a minute, leaving the T7 DNA in the supernatant.  Then we added ddH20, TAQ buffer, dNTPs, the forward and reverse minor capsid protein primers, the template DNA from the supernatant, and TAQ polymerase in a PCR tube.  We ran 35 cycles of PCR and then froze it.  To make sure we isolated the T7 minor capsid protein, we ran it with gel electrophoresis and found a single band that corresponds with the the proper base pair length.  This showed that we successfully cloned the T7 minor capsid protein gene.
-[[File:5.22PCRgel.JPG|400px|center]]
-'''3. Next Steps'''
-: Our next steps are to 1) Sequence the minor capsid protein and 2) Start the mutagenesis process in order to select for smaller phage.
+:BeginPreparing for the preperation of Ghost Particles.
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