Team:BYU Provo/Notebook/Phage Purification/Fallexp/Period1/Exp/10.25T4PCR


Phage Purification September - October Notebook: Experiments

Phage Purification

10.25 T4 PCR

I) Purpose

Identify DNA sequence difference in capsid proteins between mutants.

II) Expected Outcome

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

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)

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 5 min.
Add the above reagents in the order listed to each tube.
Run PCR using the TAQ 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
add 2 microliters of dye to 4 new tubes
extract 5 microliters of each phage and put them into each of the tubes.
We put the DNA ladder in well 1, well 2 was WT 297/298, well 3 was MUT 297/298, well 4 was WT 301/302, well 5 MUT 301/302

V) Results

[[File: | thumb|none|alt=A|T4 PCR Gel2]]