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- Phage Purification
- March-April
- May-June
- July-August
- September-October
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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.
- 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)
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
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[[File: | thumb|none|alt=A|T4 PCR Gel2]]
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