|
- Phage Purification
- March-April
- May-June
- July-August
- September-October
|
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.
- 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
-
|
"
