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- Small Phage
- March-April
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- July-August
- September-October
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10.18 Cloning T7 Capsid Protein into iGEM Registry
I) Purpose
- Clone both WT and mutant T7 Capsid Protein into iGEM registry
II) Expected Outcome
- WT and mutant T7 capsid protein cloned into the iGEM registry.
III) Reagents Used
- Reagents required for cloning: dNTP, Phusion polymerase, butter, ddH2O, etc.
- Mutant phage S4, S10, S21, and L8; WT phage
IV) Procedure
1) Amplification and purification of insert (10.18)
- I. DNA purification
- * Put 5 ul of phage sample (S4, S10, S21, L8, and WT) and 45 ul of ddH20 into in PCR tubes with respective labeling.
- * Boil for 12 minutes in the PCR machine (98 Celsius).
- * Remove the tubes from the PCR machine and keep on ice until use in PCR.
- II. PCR w/ Phusion polymerase
- * To a eppendorf tube, add (for 6 PCR reactions simultaneously):
- - 210 uL of ddH2O
- - 60uL 5x Phusion buffer
- - 9uL 10mM dNTPs
- - 6uL of each primer (BI309 and BI310)
- * Mix well
- * To six new PCR tubes, labeled S4, S10, S21, L8, and WT respectively, add 2uL of template DNA from the boiled samples.
- * Add 3uL of Phusion Polymerase to the eppendorf tube (master mix).
- * Add 48uL of the master mix into each PCR tube.
- * Run 35 cycles with temperatures of 95 C, 60 C, and 72 C with an extension time of 2 minutes.
- * Leave overnight in the freezer.
- III. Low melt gel electrophoresis (10.19)
- * Add 75 ml of 1X TAE buffer and 0.75 grams of low melt agarose to an Erlenmeyer flask. Put into the microwave for about 90 seconds or until the agarose is completely dissolved. 1 drop of ethidium bromide was then added and mixed with the gel.
- * Pour the liquid onto the gel bed and let it cool. Remember to insert a sample comb.
- * The gel allowed to sit for overnight in the fridge to solidify.
- * Move the gel into the proper orientation (DNA runs towards the red electrode) in the gel box, cover the gel with 1X TAE buffer and load 8 ul of each of the PCR products with 3 ul of loading dye mixed in. Add a DNA ladder as a reference. Turn on the power supply and run at 80 volts.
2) Repeat of the amplification and purification of insert (10.20)
- Protocols of this experiment were similar to that of 1) Amplification and purification of insert (10.18), except for the following differences
- 10uL of phage and 40uL of ddH2O was used in the boiling process
- extention time was changed to 3 miinutes
- a regular gel was used to verify PCR product
- 3uL of product and 3uL of loading dye was used per well in the gel.
V) Results
1) Amplification and purification of insert
- Only WT and S10 showed bands at approximately 1.1k bp. This experiment needs to be repeated
VI) Conclusion
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