Team:BYU Provo/Notebook/Phage Purification/Winterexp/Period1/Exp/6.5 Bacteria Cultures

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Revision as of 03:15, 6 June 2013

Phage Purification May - June Notebook: Experiments

6.5 Bacteria Cultures

I) Purpose

Grow bacteria for later use in growing phage.

II) Expected Outcome

Viable liquid cultures of both W3110 and BL21

III) Reagants Used

W3110 and BL21 freezer stock

LB

IV) Actual Procedure

Infect 45 mL of LB with 2 loop-fulls of bacterial freezer stock and let sit at 37^◦ C for 1 hour.

Infect 4 mL of LB with 1 mL of liquid culture prepared in previous step.

Let both liquid cultures sit overnight at 37^◦ C.

V) Results

A

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-<font size="5"> '''6.3 CsCl Gradient Phage Purification''' </font>
+<font size="5"> '''6.5 Bacteria Cultures''' </font>
 <br>
 '''I) Purpose'''
-: Further purify the phage to a high level of purification.
+: Grow bacteria for later use in growing phage.
 '''II) Expected Outcome'''
-: Purified and viable phage will be extracted from the CsCl gradient.
+: Viable liquid cultures of both W3110 and BL21
 '''III) Reagants Used'''
-: T4 and T7 purified phage from [[Team:BYU_Provo/Notebook/Phage_Purification/Winterexp/Period1/Exp/5.26 PEG Purification|5.26 PEG Purification]]
+: W3110 and BL21 freezer stock
-: CsCl
+: LB
-: phage suspension buffer
 '''IV) Actual Procedure'''
-: Create different concentrations of CsCl solutions to create a gradient.
+: Infect 45 mL of LB with 2 loop-fulls of bacterial freezer stock and let sit at 37<sup>◦</sup> C for 1 hour.
-:: Add 2.46 g of CsCl to 6 ml of phage suspension buffer to create a 1.3 g/ml density gradient.
+: Infect 4 mL of LB with 1 mL of liquid culture prepared in previous step.
-:: Add 4.1 g of CsCl to 6 ml of phage suspension buffer to create a 1.5 g/ml density gradient.
+: Let both liquid cultures sit overnight at 37<sup>◦</sup> C.
-:: 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 had difficulty seeing the distinct bands to extract the phage.  We will have to rerun the experiment.
+: A
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