"

From 2013.igem.org

You do not have permission to edit this page, for the following reason:

You can view and copy the source of this page:

{{TeamBYUProvo}} <br> {| width="100%" | colspan="3" | <font color="#333399" size="5" font face="Calibri"> '''Phage Purification July - August Notebook: Experiments'''</font> <br> <br> |- valign="top" | style="width: 18%; background-color: transparent;"| <font color="#333399" size="3" font face="Calibri"> : [[Team:BYU_Provo/Phage_Purification|Overview]] : [[Team:BYU Provo/Notebook/Phage_Purification/Winterexp|March-April]] : [[Team:BYU Provo/Notebook/Phage_Purification/Springexp|May-June]] : [[Team:BYU Provo/Notebook/Phage_Purification/Summerexp|July-August]] : [[Team:BYU Provo/Notebook/Phage_Purification/Fallexp|September-October]] </font> | style="width: 82%; background-color: transparent;"| <font face="Calibri" size="3"> <font size="5"> '''8.7 CsCl Gradient''' </font> <br> '''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. : 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^◦ C. : Repeat previous step three more times. : Remove purified phage from dialysis tubing and store in 4^◦ C. '''V) Results''' : 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. [[File:Phage810_1.jpg | thumb|none|alt=]] [[File:Phage810_2.jpg | thumb|none|alt=]] </font> |} {{TeamBYUProvoFooter}}

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