iGEM 2017 — Team McMaster_II

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:McMaster II/Notebook

[![](http://static1.squarespace.com/static/578a9a5cc534a56f885f0fc2/t/588d8a6c46c3c4023d83261a/1503246075934/)](https://2017.igem.org/Team%3AMcMaster_II)

MENU

**Loading...**

![Mcmaster](https://static.igem.org/mediawiki/2017/f/f1/HeaderImage_McMaster_II.jpeg)

# NoteBook

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)


[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:McMaster II/Experiments

[![](http://static1.squarespace.com/static/578a9a5cc534a56f885f0fc2/t/588d8a6c46c3c4023d83261a/1503246075934/)](https://2017.igem.org/Team%3AMcMaster_II)

MENU

**Loading...**

![Mcmaster](https://static.igem.org/mediawiki/2017/f/f1/HeaderImage_McMaster_II.jpeg)

# Experiments

## Transformation Of E. Coli

### Materials

* LB Media
* LB Plate (with antibiotic of choice)
* Ice
* DH5α or MG1655 *E. coli*

### Procedure:

1. Get Ice and set water bath to 42°C
2. Thaw *E. coli*  on ice for at least 15 minutes
3. Pipette 15 uL of ligation reaction or 1 uL of mini-prep sample into 50 uL of bacterial cells
4. Swirl the contents gently and store on ice for 30 minutes
5. Heat shock for 75 seconds in 42°C. DO NOT SHAKE!
6. Chill on ice for 2 minutes
7. Add in 600 uL of LB media and incubate at 37°C for 1 hour
8. Plate 200 uL onto LB agar plate with the appropriate antibiotic using a streaker
9. After a minimum of 12 hours, proceed to picking colonies and glycerol stocks protocol

## Picking Colonies and Glycerol Stocks

### Procedure:

1. Prepare work area and wipe it down with 70% ethanol
2. Obtain your bacterial plate(s) with the colony of interest on them
3. Using a serological pipette, dispense 5 mL of liquid culture medium (LB) into the snap cap culture tubes. One tube per colony. Then add in 5uL of corresponding antibiotic
4. Label the tubes with the plasmid and colony number
5. Using a pipette tip, gently pick a single colony form the plate and inoculate the corresponding culture tube. Drop the pipette tip in.
6. Cap the tubes to the first stop
7. Rack the culture tubes at an angle in test tube racks, and incubate in a shaking incubator at 37°C and 250 rpm.
8. Incubate for at least 12 hours, but do not exceed 18 hours
9. After overnight bacterial culture growth, add 500 uL of the culture to 500 uL of 50% glycerol in 2 mL cryo-vials and gently mix.
10. Freeze the glycerol stock tube at -80°C
11. Proceed to Mini-Prep protocol after making glycerol stocks

## Plasmid Mini-Prep

### Materials

* GeneAid Miniprep Kit
* Bacterial Cell Culture

### Procedure:

1. Spin down overnight cultures at 2000 rpm for 10 minutes
2. Discard supernatant. Do not disturb cell pellet
3. Resuspend in 500 uL of distilled water
4. Transfer to 1.5 mL microcentrifuge tube and spin at 16000\*g for 1 minute. Discard supernatant
5. Add 200 uL PD1 Buffer (with RNase A) to the tube and resuspend the pellet by vortexing
6. Add 200 uL PD2 Buffer to the tube. Invert 10 times to gently mix
7. Let stand at room temperature for at least 2 minutes, but do not exceed 5 minutes
8. Add 300 uL PD3 Buffer to the tube. Invert 10 times to mix
9. Centrifuge at 16000\*g for 3 minutes
10. Place a PD column in a 2 mL collection tube
11. Carefully transfer the supernatant from Step 9 without transferring any of the white precipitate
12. Centrifuge at 16000\*g for 30 seconds and discard the flow-through
13. Add 400 uL W1 Buffer to the tube and centrifuge at 16000\*g for 30 seconds. Discard the flow-through
14. Add 600 uL Wash Buffer (with ethanol) to the tube and centrifuge at 16000\*g for 30 seconds. Discard the flow-through
15. Centrifuge the column and collection tube at 16000\*g for 3 minutes to dry the column
16. Transfer the column into a new 1.5 mL microcentrifuge tube
17. Add 50 uL of ddH2O into the centre of the column matrix
18. Let stand for at least 2 minutes for absorption
19. Centrifuge at 16000\*g for 2 minutes to elute the purified DNA.
20. Use a spectrophotometer to measure the DNA concentration of the purified sample

## Colony PCR with Taq Mastermix

### Procedure:

1. Pick a colony into 20 uL ddH 2 O and ensure all of the cells have been transferred
2. Create the following reaction for each colony you wish to amplify:

| Reagent | Amount |
| --- | --- |
| **PCR Mastermix (5X)** | 2 |
| **Forward** | 0.1 |
| **Primer** |  |
| **50uM Reverse Primer** | 0.1 |
| **Water** | 7.3 |
| **Subtotal** | 9.5uL |
| **Colony Template** | 0.5 |
| **Total** | 10 uL |

3. Run with PCR the following protocol:

| Reagent | Temperature (°C) | Time | Cycles |
| --- | --- | --- | --- |
| **Enzyme Activation** | 95 | 15 min | 1X |
| **Denature** | 95 | 30 sec | 35-40X |
| **Anneal** | T m | 30 sec |  |
| **Extend** | 72 | See note below |  |
| **Final Extension** | 72 | 7 min | 1X |
| **Hold** | 4 | Hold | 1X |

Note: The extension time should be 1min/kb. Round up for this step. Ex: For a 3.6 kb construct, use a 4 min extension time. It is helpful to be generous with the extension time

4. Proceed to Agarose Gel Electrophoresis Protocol to analyze results of PCR

## Agarose Gel Electrophoresis

### Procedure:

1. Prepare a 100 mL of TAE buffer and add in agarose (concentration from 0.8 – 3% w/v) in a
   250 mL flask and mx it thoroughly. Place the flask in the microwave, heat until the solution
   is completely clear. The agarose concentration depends on the band size of interest (2kb –
   10kb → 1%; 1kb – 2kb → 1.5%; 200bp – 1kb → 2%; 40bp – 200bp → 3%
2. When the gel does not burn your hand anymore, add 5 uL of RedSafe Nucleic Staining
   solution (20000X) to the agarose solution. Swirl the flask gently to mix the solution and
   avoid forming bubbles (5uL for 100mL, 10uL for 200mL)
3. Pour it into the gel tray until the comb teeth are immersed about ¼ - ½ into the agarose
4. Allow the agarose gel to cool until solidified. This usually takes 20 minutes
5. Add adequate amount of 6X loading dye to DNA samples to have a final concentration of
   1X. Load samples on the gel
6. Perform electrophoresis: 120 V for 45 minutes
7. Detect the bands under UV illumination

## Gel Extraction

### Procedure:

1. Excise the agarose gel slice containing the relevant DNA fragments. Remove any excess
   agarose to minimize size of gel slice
2. Transfer up to 300 mg of the gel slice to a 1.5 mL microcentrifuge tube
3. Add 500 uL of DF Buffer to the same and mix by vortex
4. Incubate at 50-60°C for 10-15 minutes to ensure the gel slice is fully dissolved. Invert every
   2-3 minutes.
5. Cool the dissolved sample mixture to room temperature
6. Place the DF column in a 2 mL Collection Tube
7. Transfer 800 uL of the sample mixture to the DF column
8. Centrifuge at 16000\*g for 30 seconds. Discard flow-through
9. Add 400 uL of W1 Buffer into the DF column
10. Centrifuge at 16000\*g for 30 seconds. Discard the flow-through
11. Add 600 uL of Wash Buffer (with ethanol) into the DF column
12. Let stand for 1 minute at room temperature
13. Centrifuge at 16000\*g for 30 seconds. Discard the flow-through
14. Centrifuge at 16000\*g for 3 minutes to dry the column matrix
15. Transfer the dried DF column to a new 1.5 mL microcentrifuge tube
16. Add 30 uL of ddH2O into the center of the column matrix
17. Let stand for 2 minutes to ensure water is completely absorbed
18. Centrifuge at 16000\*g for 2 minutes to elute the purified DNA
19. Use a spectrophotometer to measure the DNA concentration of the purified sample

## Colony PCR with Taq Mastermix

### Procedure for Digestion:

1. Determine what digestions need to be done and the enzymes required for these digestions
2. The amount of DNA necessary for digestion is 500 ng. Calculate the volume of DNA
   required based on its concentration.
3. Determine the optimal buffer for the double digest. For EcoRI/SpeI digestion, use 10X
   CutSmart Buffer. For XbaI/PstI digestion, use 10X Buffer 3.1. For EcoRI/PstI digestion,
   use 10X Buffer 2.1.
4. Add the following materials into a 1.5 mL microcentrifuge tube:

| Reagent | Volume (uL) |
| --- | --- |
| **DNA (500ng)** | Calculated volume |
| **Enzyme 1** | 1 |
| **Enzyme 2** | 1 |
| **Optimal** | 5 |
| **Buffer** |  |
| **Water** | Up to 50 uL total volume |
| **Total** | 50uL |

5. Run with PCR the following protocol:
6. Spin down the microcentrifuge tube to ensure all reagents are to mixed
7. Incubate at 37°C for at least 1 hour.
8. Proceed to Digestion Cleanup.

### Procedure for Digestion Cleanup:

1. Transfer 50 uL of digestion product to a 1.5 mL microcentrifuge tube
2. Add 5 volumes of DF Buffer (250 uL) to 1 volume of sample and mix by vortex
3. Place a DF column in a 2 mL collection tube.
4. Transfer the sample mixture to the DF column
5. Centrifuge at 16000\*g for 30 seconds. Discard the flow-through
6. Add 600 uL of Wash Buffer (with ethanol) into the center of the DF column
7. Let stand for 1 minute at room temperature
8. Centrifuge at 16000\*g for 30 seconds. Discard the flow-through
9. Centrifuge at 16000\*g for 3 minutes to dry the column matrix
10. Transfer the dried DF column to new 1.5 mL microcentrifuge tube
11. Add 20 uL of ddH2O into the center of the column matrix
12. Let stand for at least 2 minutes to ensure absorption of water
13. Centrifuge at 16000\*g for 2 minutes to elute the purified DNA
14. Proceed to Ligation

### Procedure for Ligation:

1. Obtain the digested inserts and backbones of interest
2. Add the following reagents to a 1.5 mL microcentrifuge tube in the following amounts:

| Reagent | Volume (uL) |
| --- | --- |
| **Insert 1** | 3 |
| **Insert 2** | 3 |
| **Backbone** | 2 |
| **10X Ligase Buffer** | 1 |
| **Ligase** | 1 |
| **Water** | Up to 10 uL total volume |
| **Total** | 10uL |

3. Incubate the ligations at 37°C for at least 30 minutes. However, the longer the incubation,
   the higher efficiency for ligation
4. Transform the ligation products into E. coli. Please refer to Transformation protocol.

## PCR Cleanup Protocol

### Procedure

1. Transfer up to 100 uL of reaction product to a 1.5 mL microcentrifuge tube
2. Add 5 volumes of DF Buffer to 1 volume of sample and mix by vortex
3. Place a DF column in a 2 mL collection tube.
4. Transfer the sample mixture to the DF column
5. Centrifuge at 16000\*g for 30 seconds. Discard the flow-through
6. Add 600 uL of Wash Buffer (with ethanol) into the center of the DF column
7. Let stand for 1 minute at room temperature
8. Centrifuge at 16000\*g for 30 seconds. Discard the flow-through
9. Centrifuge at 16000\*g for 3 minutes to dry the column matrix
10. Transfer the dried DF column to new 1.5 mL microcentrifuge tube
11. Add 20 uL of ddH2O into the center of the column matrix
12. Let stand for at least 2 minutes to ensure absorption of water
13. Centrifuge at 16000\*g for 2 minutes to elute the purified DNA

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)

[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:McMaster II/Results

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3AMcMaster_II)

MENU

[HOME](https://2017.igem.org/Team%3AMcMaster_II)

TEAM

[Team](https://2017.igem.org/Team%3AMcMaster_II/Team)
[Collaborations](https://2017.igem.org/Team%3AMcMaster_II/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3AMcMaster_II/Description)
[Design](https://2017.igem.org/Team%3AMcMaster_II/Design)
[Experiments](https://2017.igem.org/Team%3AMcMaster_II/Experiments)
[Notebook](https://2017.igem.org/Team%3AMcMaster_II/Notebook)
[InterLab](https://2017.igem.org/Team%3AMcMaster_II/InterLab)
[Contribution](https://2017.igem.org/Team%3AMcMaster_II/Contribution)
[Model](https://2017.igem.org/Team%3AMcMaster_II/Model)
[Results](https://2017.igem.org/Team%3AMcMaster_II/Results)
[Demonstrate](https://2017.igem.org/Team%3AMcMaster_II/Demonstrate)
[Improve](https://2017.igem.org/Team%3AMcMaster_II/Improve)
[Attributions](https://2017.igem.org/Team%3AMcMaster_II/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3AMcMaster_II/Parts)
[Basic Parts](https://2017.igem.org/Team%3AMcMaster_II/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3AMcMaster_II/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3AMcMaster_II/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3AMcMaster_II/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3AMcMaster_II/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3AMcMaster_II/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3AMcMaster_II/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3AMcMaster_II/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3AMcMaster_II/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3AMcMaster_II/Hardware)
[Measurement](https://2017.igem.org/Team%3AMcMaster_II/Measurement)
[Model](https://2017.igem.org/Team%3AMcMaster_II/Model)
[Plant](https://2017.igem.org/Team%3AMcMaster_II/Plant)
[Software](https://2017.igem.org/Team%3AMcMaster_II/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=McMaster_II)

# McMaster\_II

# Results

Here you can describe the results of your project and your future plans.

##### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

##### You should also describe what your results mean:

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember all measurement and characterization data must be on part pages in the Registry.
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

##### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

##### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)

[![](https://static.igem.org/mediawiki/2017/b/bf/HQ_menu_logo.jpg)](https://2017.igem.org)