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


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Phage Purification September - October Notebook: Experiments

Phage Purification

10.21 T4 PCR

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.
2.5 microL Thermo Pol. buffer (for high fidelity TAQ)
1 microL primers (B1 297/B1 298) (B1 302/B1 303)
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 (B1 311/B1 312) (B1 313/B1 314)
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 99 C for 12 min.
Add the above reagents in the order listed to each tube.
Run PCR using the TAQ protocol and the phusion protocol
Run in 1% gel
100 mL 1x TAE buffer
1 g regular agarose
heat until dissolved completely
cool and add 2 drops ethidium bromide
pour with 14 well mold and allow to solidify

V) Results

T4 PCR Gel1
T7 Second Gradient