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

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: [[Team:BYU_Provo/Phage_Purification|Overview]]
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: <u> '''Phage Purification''' </u> </font>
: [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
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: Fill the remaining space in the tube with phage suspension buffer to the top.
: 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.
: Centrifuge at 26500 rpms (100,000 g) for 2.5 hours.
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: Extract using a needle and puncturing the side of the tube, placing the needle underneath the band.
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: Place phage in dialysis tubing and place in flask with 1 L of phage suspension buffer for 30 minutes at 4<sup>◦</sup> C.
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: Repeat previous step three more times.
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: Remove purified phage from dialysis tubing and store in 4<sup>◦</sup> C.
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'''V) Results'''
'''V) Results'''
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: The phage banded all at the same 1.3 spot in the gradient.  Because of this, we were unable to purify small phage from the wild type.
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: 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.
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Latest revision as of 00:36, 28 September 2013


Phage Purification July - August Notebook: Experiments



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

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.


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