Team:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry
From 2013.igem.org
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:: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix). | :: * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix). | ||
:: * Add 48uL of the master mix into each PCR tube. | :: * Add 48uL of the master mix into each PCR tube. | ||
- | :: * Run 35 cycles with temperatures of 95 C, | + | :: * Run 35 cycles with temperatures of 95 C, 60 C, and 72 C with an extension time of 2 minutes. |
:: * Leave overnight in the freezer. | :: * Leave overnight in the freezer. | ||
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:: * The gel allowed to sit for overnight in the fridge to solidify. | :: * The gel allowed to sit for overnight in the fridge to solidify. | ||
:: * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts. | :: * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts. | ||
+ | |||
+ | 2) Repeat of the amplification and purification of insert (10.20) | ||
+ | : Protocols of this experiment were similar to that of 1) Amplification and purification of insert (10.18), except for the following differences | ||
+ | * 10uL of phage and 40uL of ddH2O was used in the boiling process | ||
+ | * extention time was changed to 3 miinutes | ||
+ | * a regular gel was used to verify PCR product | ||
+ | * 3uL of product and 3uL of loading dye was used per well in the gel. | ||
'''V) Results''' | '''V) Results''' | ||
+ | 1) Amplification and purification of insert | ||
+ | : Only WT and S10 showed bands at approximately 1.1k bp. This experiment needs to be repeated | ||
+ | |||
'''VI) Conclusion''' | '''VI) Conclusion''' |
Revision as of 16:03, 21 October 2013
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10.18 Cloning T7 Capsid Protein into iGEM Registry
I) Purpose
II) Expected Outcome
III) Reagents Used
IV) Procedure 1) Amplification and purification of insert (10.18)
2) Repeat of the amplification and purification of insert (10.20)
V) Results 1) Amplification and purification of insert
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