Team:BYU Provo/Notebook/Phage Purification/Fallexp/Period1/Exp/10.21T4PCR

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Phage Purification September - October Notebook: Experiments



Phage Purification
March-April
May-June
July-August
September-October

10.16 T7 EM


I) Purpose

Identify DNA sequence difference in capsid proteins between mutants. Also, to submit DNA sequences of capsids to the iGEM registry.

II) Expected Outcome

PCR products of the 2 T4 capsid protein genes for wild type and mutant phages.

III) Reagants Used

2 microL T4 wild type or mutant phages in each.
TAQ PCR
2.5 microL Thermo Pol. buffer (for high fidelity TAQ)
1 microL Forward (B1297) and 1 microL Reverse (B1298) Primers
17 microL ddH20
1 microL 10 mM dNTP's
.5 microL Polymerase (TAQ)
Phusion PCR
10 microL HF Phusion Buffer
1.5 microL dNTP's
1.5 microL of each primer, forward and reverse.
32.5 microL ddH20
1 microL Phusion polymerase

We set up the primers to isolate the small and large capsid protein genes separately in Phusion for cloning the genes into the registry. (4 samples) The TAQ was run to amplify and sequence the capsid proteins using two sets of different primers. (4 samples)

IV) Actual Procedure

Put 2 microL phage in an eppendorf tube. Incubate at 42 C for 12 min.
Add the above reagents in the order listed to each tube.

V) Results


A
T4 PCR Gel1
A
T7 Second Gradient