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

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Revision as of 21:07, 23 September 2013

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.

V) Results

The phage banded ll at 1.3, allowing for no separation between wild type and mutant. The gradient will have to be run again. No phage was extracted or dialyzed.

@@ Line 41: / Line 41: @@
 '''III) Reagants Used'''
-: T4 mutant phage
+: T7 mutant phage
 : CsCl
 : dialysis tubing
@@ Line 65: / Line 65: @@
 : 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 three more times.
-: Remove purified phage from dialysis tubing and store in 4<sup>◦</sup> C.
 '''V) Results'''
-: The phage banded all at the same 1.5003 spot in the gradient. We will have to assume that there is mutated phage below this upper band.
+: The phage banded ll at 1.3, allowing for no separation between wild type and mutant. The gradient will have to be run again. No phage was extracted or dialyzed.
 [[File:Phage810_1.jpg | thumb|none|alt=]]
 [[File:Phage810_2.jpg | thumb|none|alt=]]