Team:BYU Provo/Notebook/SmallPhage/Fallexp/10.18 Cloning T7 Capsid Protein into iGEM Registry

Small Phage

March-April

May-June

July-August

September-October

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, WT, and control 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.

3) Restriction digest (10.21)

* Followed procedure for PCR cleanup of WT, S4, S10, S21, and L8.

* For each PCR product (WT, S4, S10, S21, and L8), we set up an individual tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of PCR product, and 2ul of each restriction enzyme.

* We also made a vector digestion tube with 14ul of water, 5ul of 10X NEB buffer, 0.5ul 100X BSA, 30ul of the vector digestion, and 2ul of each restriction enzyme.

* The six tubes were placed in the 37C incubator overnight.

4) Low-melt Gel for Purification of Sticky-ended Insert/Vector (10.22)

* Ran a low-melt gel for each of the tubes in step 3.

* Cut out each bright band and placed them in an eppendorf tube.

* Left them in the freezer overnight.

5) Ligation (10.23)

* Centrifuged the eppendorf tubes from step 4 to separate the vector or insert from the gel.

* Created 5 ligation reactions for each phage (WT, S4, S10, S21, and L8) with 6.5ul water, 1.5ul 10X ligase buffer, 1ul T4 DNA ligase, 3ul vector, and 3ul insert.

6) Transformation (10.23)

* Added 2ul of ligation mix to 25ul of competent cells. Vortexed the tubes briefly and placed them on ice for 10 minutes.

* Heat shocked at 42C for 1 minute and then placed tubes back on ice for 10 minutes.

* Added 0.5mL of plain LB to the reactions and incubated at 37C for 1 hour.

* Plated 100ul of cells on CAM plates and incubated at 37C overnight.

7) Colony PCR and Overnights for Vector Purification (10.24)

* Our transformations only had WTa, WTb, S4, S10a, S10b, S10c, S21, L8a, and L8b (a,b,c designates multiple colonies from the same transformation.

* For each colony, we picked it with a pipet tip, dipped it in 50ul of water in a PCR tube, and placed the tip in a test tube of 5mL of LB (These tubes were placed in the 37C incubator as overnights).

* Boil each PCR tube for 5 minutes.

* Set up a PCR reaction for each PCR tube by adding 2.5ul Standard 10X reaction buffer, 0.5ul 10mM dNTP's, 0.5ul of each primer, 0.5ul Taq DNA polymerase, and 2ul of boiled colony sample.

* Run PCR for 25 cycles.

* Run a gel with 5ul of each PCR product to check for proper size.

8) Plasmid Cleanup (10.25)

* Followed procedure for plasmid cleanup for WT, S4, S10, and L8.

* Mailed 10ul of each plasmid to iGEM!

Monday: Restriction digest Tuesday: Low-melt gel for purification of sticky-ended insert/vector (Jade) Wednesday: Ligation and transformation Thursday: Redid plating for transformation and started overnights of each colony and ran gel Friday: Cleaned up the plasmid and sent it in

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

The cloning worked for WT, S4, S10, and L8. We submitted all these parts to the registry.