Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/8.7CsClGradient

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Phage Purification July - August Notebook: Experiments

8.7 CsCl Gradient

I) Purpose

Further purify the phage to a high level of purification.

II) Expected Outcome

Purified and viable phage will be extracted from the CsCl gradient.

III) Reagants Used

T7 mutant phage

CsCl

dialysis tubing

phage suspension buffer

IV) Actual Procedure

Create different concentrations of CsCl solutions to create a gradient.

Add 1.64 g of CsCl to 4 mL of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 2.3466 g of CsCl to 4 mL of phage suspension buffer to create a 1.43 g/ml density gradient.

Add 3.6840 g of CsCl to 6 mL of phage suspension buffer to create a 1.45 g/ml density gradient.

Add 3.8483 g of CsCl to 6 mL of phage suspension buffer to create a 1.47 g/ml density gradient.

Add 4.0978 g of CsCl to 6 mL of phage suspension buffer to create a 1.5003 g/ml density gradient.

Add 2.7362 g of CsCl to 4 mL of phage suspension buffer to create a 1.5011 g/ml density gradient.

Add 2.7406 g of CsCl to 4 mL of phage suspension buffer to create a 1.5019 g/ml density gradient.

Add 4.1159 g of CsCl to 6 mL of phage suspension buffer to create a 1.5025 g/ml density gradient.

Add 2.7489 g of CsCl to 4 mL of phage suspension buffer to create a 1.5034 g/ml density gradient.

Add 4.1283 g of CsCl to 6 mL of phage suspension buffer to create a 1.5040 g/ml density gradient.

Add 4.9222 g of CsCl to 6 mL of phage suspension buffer to create a 1.6 g/ml density gradient.

Add 5.7549 g of CsCl to 6 mL of phage suspension buffer to create a 1.7 g/ml density gradient.

Layer the gradient into two centrifuge tubes, using half of the total volume created for each tube. For example, add 2 mL of 1.3 density to one tube and the other 2 mL to another tube.

Fill the remaining space in the tube with phage suspension buffer to the top.

Centrifuge at 26500 rpms (100,000 g) for 2.5 hours.

Extract using a needle and puncturing the side of the tube, placing the needle underneath the band.

Place phage in dialysis tubing and place in flask with 1 L of phage suspension buffer for 30 minutes at 4^◦ C.

Repeat previous step three more times.

Remove purified phage from dialysis tubing and store in 4^◦ C.

V) Results

The phage banded all at the same spot in the gradient. Because of this, we were unable to purify small phage from the wild type.

@@ Line 4: / Line 4: @@
 {| width="100%"
-| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Phage Purification May - June Notebook: Experiments'''</font>
+| colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Phage Purification July - August Notebook: Experiments'''</font>
 <br>
@@ Line 14: / Line 14: @@
 <font color="#333399" size="3" font face="Calibri">
-: [[Team:BYU_Provo/Small_Phage|Overview]]
+: [[Team:BYU_Provo/Phage_Purification|Overview]]
 : [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
@@ Line 30: / Line 30: @@
 <font face="Calibri" size="3">
-<font size="5"> '''6.24 CsCl Gradient''' </font>
+<font size="5"> '''8.7 CsCl Gradient''' </font>
 <br>
@@ Line 41: / Line 41: @@
 '''III) Reagants Used'''
-: T7 purified phage
+: T7 mutant phage
 : CsCl
 : dialysis tubing
@@ Line 50: / Line 50: @@
 : Create different concentrations of CsCl solutions to create a gradient.
-:: Add 1.64 g of CsCl to 4 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
+:: Add 1.64 g of CsCl to 4 mL of phage suspension buffer to create a 1.3 g/ml density gradient.
-:: Add 4.10 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.
+:: Add 2.3466 g of CsCl to 4 mL of phage suspension buffer to create a 1.43 g/ml density gradient.
-:: Add 4.92 g of CsCl to 6 ml of phage suspension buffer to create a 1.6 g/ml density gradient.
+:: Add 3.6840 g of CsCl to 6 mL of phage suspension buffer to create a 1.45 g/ml density gradient.
-:: Add 5.76 g of CsCl to 6 ml of phage suspension buffer to create a 1.7 g/ml density gradient.
+:: Add 3.8483 g of CsCl to 6 mL of phage suspension buffer to create a 1.47 g/ml density gradient.
-: Layer a centrifuge tube with 3 mL 1.7 g/mL, 3 mL 1.6 g/mL, 3 mL of 1.5 g/mL, and then 2 mL of 1.3 g/mL.
+:: Add 4.0978 g of CsCl to 6 mL of phage suspension buffer to create a 1.5003 g/ml density gradient.
-: Layer T7 on top of the gradient in separate tubes(as much as is available).
+:: Add 2.7362 g of CsCl to 4 mL of phage suspension buffer to create a 1.5011 g/ml density gradient.
+:: Add 2.7406 g of CsCl to 4 mL of phage suspension buffer to create a 1.5019 g/ml density gradient.
+:: Add 4.1159 g of CsCl to 6 mL of phage suspension buffer to create a 1.5025 g/ml density gradient.
+:: Add 2.7489 g of CsCl to 4 mL of phage suspension buffer to create a 1.5034 g/ml density gradient.
+:: Add 4.1283 g of CsCl to 6 mL of phage suspension buffer to create a 1.5040 g/ml density gradient.
+:: Add 4.9222 g of CsCl to 6 mL of phage suspension buffer to create a 1.6 g/ml density gradient.
+:: Add 5.7549 g of CsCl to 6 mL of phage suspension buffer to create a 1.7 g/ml density gradient.
+: Layer the gradient into two centrifuge tubes, using half of the total volume created for each tube.  For example, add 2 mL of 1.3 density to one tube and the other 2 mL to another tube.
 : Fill the remaining space in the tube with phage suspension buffer to the top.
 : Centrifuge at 26500 rpms (100,000 g) for 2.5 hours.
 : Extract using a needle and puncturing the side of the tube, placing the needle underneath the band.
 : Place phage in dialysis tubing and place in flask with 1 L of phage suspension buffer for 30 minutes at 4<sup>◦</sup> C.
-: Repeat previous step two more times.
+: Repeat previous step three more times.
 : Remove purified phage from dialysis tubing and store in 4<sup>◦</sup> C.
 '''V) Results'''
-: We were able to successfully extract phage from the CsCl.  Dr. Grose explained that the band which we had previously thought to be the bacterial debris was probably the purified phage.  We removed the phage from this band and purified it through dialysis.  Next class we will be performing a titer to determine the concentration of the phage.
+: The phage banded all at the same spot in the gradient.  Because of this, we were unable to purify small phage from the wild type.
 </font>