Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/5.26 PEG Purification

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Latest revision as of 00:26, 28 September 2013

Phage Purification May - June Notebook: Experiments

Phage Purification

5.26 PEG Purification

I) Purpose

The first step in the phage purification process. Purify phage from bacterial debris.

II) Expected Outcome

Phage will be purified from the bacterial debris to be further purified in a CsCl gradient.

III) Reagants Used

phage suspension buffer

10 mM Tris-HCl

100 mM NaCl

10 mM MgCl₂

DNase I

RNase A

chloroform

NaCl powder

PEG 8000

T4 and T7 bacterial lysate

IV) Actual Procedure

Add 185 µL DNase and 25 µL RNase to T4 and T7 bacterial lysates.

Add .1 mL chloroform to complete lysis and let the preparations sit for 30 minutes at room temperature.

Dissolve 1.465 g of solid NaCl and let cool at 4^◦ C for 1 hour.

Centrifuge the suspensions at 6500 rpms (7000 g) for 10 minutes at 4^◦ C. Remove supernatant and put in clean flasks.

Dissolve 4.05 g PEG 8000 and let sit at 4^◦ C for 1 hour.

Centrifuge the suspensions at 8000 rpms (10000 g) for 15 minutes at 4^◦ C. Discard the supernatant.

Turn the centrifuge bottles over to let sit for 5 minutes to drain off any remaining supernatant.

Suspend the pellets in .675 mL phage suspension buffer and let sit overnight at 4^◦ C.

V) Results

We were able to purify phage to a point that it can now be used in the CsCl gradient. We finished with phage dissolved in phage suspension buffer. We will be using this solution in a later class to further purify in a CsCl gradient.

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-: [[Team:BYU_Provo/Small_Phage|Overview]]
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+: <u> '''Phage Purification''' </u> </font>
 : [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]]
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 '''III) Reagants Used'''
 :
-: CsCl
 : phage suspension buffer
+:: 10 mM Tris-HCl
+:: 100 mM NaCl
+:: 10 mM MgCl<sub>2</sub>
+: DNase I
+: RNase A
+: chloroform
+: NaCl powder
+: PEG 8000
+: T4 and T7 bacterial lysate
 '''IV) Actual Procedure'''
-: Create different concentrations of CsCl solutions to create a gradient.
+: Add 185 µL DNase and 25 µL RNase to T4 and T7 bacterial lysates.
-:: Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
+: Add .1 mL chloroform to complete lysis and let the preparations sit for 30 minutes at room temperature.
-:: Add 4.1 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.
+: Dissolve 1.465 g of solid NaCl and let cool at 4<sup>◦</sup> C for 1 hour.
-:: Add 4.92 g of CsCl to 6 ml of phage suspension buffer to create a 1.6 g/ml density gradient.
+: Centrifuge the suspensions at 6500 rpms (7000 g) for 10 minutes at 4<sup>◦</sup> C.  Remove supernatant and put in clean flasks.
-: Layer two centrifuge tubes with 2 mL 1.6 g/mL, 3 mL of 1.5 g/mL, and then 3 mL of 1.3 g/mL.
+: Dissolve 4.05 g PEG 8000 and let sit at 4<sup>◦</sup> C for 1 hour.
-:Layer T4 and T7 on top of the gradient in separate tubes.
+: Centrifuge the suspensions at 8000 rpms (10000 g) for 15 minutes at 4<sup>◦</sup> C. Discard the supernatant.
-:Fill the remaining space in the tube with phage suspension buffer to 3-5 mL from the top.
+: Turn the centrifuge bottles over to let sit for 5 minutes to drain off any remaining supernatant.
-:Centrifuge at 26500 rpms for 2.5 hours.
+: Suspend the pellets in .675 mL phage suspension buffer and let sit overnight at 4<sup>◦</sup> C.
-:Leave overnight in the refrigerator.
 '''V) Results'''
-: After centrifugation, we were able to see a distinct band of phage in both the T4 and T7 gradients.  The T7 band was significantly lower in the tube than the T4 band.  Next time we run the procedure, we will be running the T7 phage through a higher concentrated gradient.  After leaving the gradients overnight in the refrigerator, the gradients mixed together and we were unable to see the distinct bands to extract the phage.  We will have to rerun the experiment.
+: We were able to purify phage to a point that it can now be used in the CsCl gradient.  We finished with phage dissolved in phage suspension buffer.  We will be using this solution in a later class to further purify in a CsCl gradient.
 </font>