Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/CsClGradientPhagePurification

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Phage Purification March - April Notebook: Experiments

6.3 CsCl Gradient Phage Purification

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

T4 and T7 purified phage from step 1

CsCl

phage suspension buffer

IV) Actual Procedure

Create different concentrations of CsCl solutions to create a gradient.

Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.

Add 4.1 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 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.

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.

Layer T4 and T7 on top of the gradient in separate tubes.

Fill the remaining space in the tube with phage suspension buffer to 3-5 mL from the top.

Centrifuge at 26500 rpms for 2.5 hours.

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.

@@ Line 35: / Line 35: @@
 '''I) Purpose'''
-: Determine the concentration of the 5.3 T7 new Phage Stock to give an estimate for starting mutagenesis
+: Further purify the phage to a high level of purification.
 '''II) Expected Outcome'''
-: As the dilution series increases, the number of plaques on the plates should decrease
+: Purified and viable phage will be extracted from the CsCl gradient.
 '''III) Reagants Used'''
-: E5.3 T7 new phage stock; LB; BL21 E. coli from 5.14 overnight
+: T4 and T7 purified phage from step 1
+: CsCl
+: phage suspension buffer
 '''IV) Actual Procedure'''
-: Performed dilution series with 5.3 T7 stock; went down to -8
+: Create different concentrations of CsCl solutions to create a gradient.
-: Add 0.5 mL of BL21 to 4 test tubes, labelled 5, 6, 7, 8
+:: Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
-: Add 20 uL of T7 new to each test tube. Phage concentration should correspond to the number on the test tube. Incubate at room temp for 20 minutes.
+:: Add 4.1 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.
-: In a 50mL centrifuge tube, combine 10mL of LB with 10mL of x2 top agar.
+:: Add 4.92 g of CsCl to 6 ml of phage suspension buffer to create a 1.6 g/ml density gradient.
-: Add 5mL of the combined solution to each of the four test tubes. Plate each tube and incubate at 37 C for 24 hours
+: 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.
+:Layer T4 and T7 on top of the gradient in separate tubes.
+:Fill the remaining space in the tube with phage suspension buffer to 3-5 mL from the top.
+:Centrifuge at 26500 rpms for 2.5 hours.
+:Leave overnight in the refrigerator.
 '''V) Results'''
-: Lots of contamination - from the plates -> this batch of LB plates have been completely disposed of.
+: 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.
-: -8 plate had 7 plaques: This gives a phage concentration estimate of approximately 7E8 particle/20uL
-: -5, -6, and -7 had overlapping plaques
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