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

Phage Purification

March-April

May-June

July-August

September-October

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 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