iGEM 2013 — Team UT_Dallas

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# Team:UT Dallas/Notebook

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## Protocols

### Ligation Protocol

- Determine insert to vector ratios
- Calculate the amount of insert needed if 50ng of vector is used (can use different amount of vector)
- In a PCR tube add the following:
  > - 50ng of vector
  > - Amount of insert based on ratios (calculated in second step)
  > - 2uL of buffer
  > - 2uL of DNA ligase
  > - Amount of water to bring total volume to 20uL
- Incubate overnight at 14 degrees Celsius

Note: We used T4 DNA ligase and buffer from NEB

### Gel Purification Protocol (QIAquick Gel Extraction Kit)

- Excise DNA fragment from the agarose gel with a clean, sharp scalpel
- Weigh the gel slice in a microcentrifuge tube.
- Add 3 volumes of Buffer QG to 1 volume of gel (100mg~100uL)
- Incubate at 50 degrees Celsius for 10 min (until the gel slice has completely dissolved)
- After the gel slice has dissolved completely, check that the color of the mixture is yellow
- Apply the sample to a QIAquick column, and centrifuge for 1 min
- > Maximum volume of the column is 800uL. For samples larger than this, simply load and spin again.
- Discard flow-through and place QIAquick column back in the same collection tube
- To wash, add 750uL of Buffer PE to column and centrifuge for 1 min.
- Discard the flow-through and centrifuge for additional 1 min. at 13,000rpm
- Place QIAquick column into a clean 1.5 mL microcentrifuge tube
- To elute DNA, add 50uL of Buffer EB to the center of the QIAquick membrane, let the column stand for 1 min. and then centrifuge the column for 1 min.

### Gel Electrophoresis Protocol

- Making a 1% agarose gel
  > - 100mL 1X TBE buffer
  > - 1g agarose
  > - microwave until agarose dissolves
  > - let mixture cool
  > - when cool add 8-10uL ethidium bromide
  > - stir gently, let cool
  > - pour into plate with comb already in place
  > - let harden
- Using the gel
  > - Add loading buffer to DNA (for 100uL DNA, add 20uL loading buffer)
  > - Load 2uL of DNA ladder into the gel
  > - Load DNA into the gel
  > - Run at 130V for 30min-1hr

  ### Digestion Protocol
- Using a microcentrifuge tube add the following:
  > - ~3000-5000 ng of DNA
  > - 10uL Buffer 4
  > - 10uL BSA
  > - 5uL of appropriate enzyme (if doing a double digest, use 5 uL of both enzymes)
  > - Amount of H2O needed to make final volume 100uL
- Incubate at 37 degrees Celsius for 1hr and 30min

Note: We used the following enzymes from NEB: EcoRI-HF, PstI-HF, SpeI, and XbaI. All of which can be double digested with each other using Buffer 4.

### Preparing LB+Appropriate Antibiotic Protocol

- 200 mL LB broth
- Autoclave
  > - Put control thermometer in H2O (from the sink)
  > - Select vented container mode (Do Not Change Program)
- Let cool to 50 degrees Celsius
- Add antibiotic (50-100 ug/mL) (10 mg total)
  > - Weigh on paper
  > - Add to 0.5 mL DI H2O
  > - Add to LB mixture when cool enough
- Store at 4 degrees Celsius

### Preparing Agar Plates Protocol (Makes 12 (15mm) Plates)

- 300 mL DI H2O + 11 g LB agar
- Autoclave
  > - Put control thermometer in H2O (from the sink)
  > - Select vented container mode (Do Not Change Program)
- Mix well after autoclaving; let cool to 50 degrees Celsius
- Add antibiotic (50 to 100 µg/mL) (15 mg total)
  > - Weigh on paper
  > - Add to 0.5 mL DI H2O
  > - Add to LB mixture when cool enough
- Plate
  > - Under flame open lids of all plates
  > - Slowly pour agar into plate, avoiding bubbles, when it touches all edges stop pouring
  > - Let sit under flames until gel solidifies
  > - Replace lids on plates
- Store upside down at 4 degrees Celsius

### Preparing Competent Cells Protocol

- Place 1 colony in 5 mL of LB (with antibiotics if appropriate) Grow overnight at 37 degrees Celsius and 200-300 rpm
- Inoculate 0.25 mL of the overnight strain into 25 mL of LB
- Shake at 37oC until the OD650 is 0.6-0.7
- Harvest cells and resuspend in 12.5 mL ice cold 0.1M MgCl2
- Harvest immediately and resuspend in 7.5 mL cold 0.1M CaCl2
- Leave on ice for 30 minutes. Harvest and resuspend in 2.5 mL cold 0.1M CaCl2
- Leave on ice for 30 minutes
- For long term storage, use 0.1M CaCl2 in 15% glycerol at step 6 and store cells at -800 degrees Celsius

Note: Harvest cells at 5000 rpm for 10 minutes at 4 degrees Celsius

### Miniprep Protocol (from QIAprep Spin Miniprep Kit)

- Harvest cells at 5400g 10 minutes 40 degrees Celsius (possibly program 1)
- Resuspend pelleted bacterial cells in 250 µL Buffer P1 and transfer to a microcentrifuge tube
- Add 250 µL Buffer P2 and mix thoroughly by inverting the tube 4-6 times
- Add 350 µL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times
- Centrifuge for 10 minutes at 13000 rpm (~17900g) in a table-top microcentrifuge
- Apply the supernatant (from step 4) to the QIA prep spin column by decanting or pipetting
- Centrifuge for 30-60 seconds. Discard the flow-through
- Wash QIA prep spin column by adding 0.75 mL Buffer PE and centrifuging for 30-60 seconds
- Discard the flow-through, and centrifuge for 1 minute to remove residual wash buffer
- To elute DNA, place the QIA prep column in a clean 1.5 mL microcentrifuge tube. Add 50 µL Buffer EB or water to the center of each QIA prep spin column, let stand for 1 minute and centrifuge for 1 minute.

### Preparing Glycerol Stock Protocol

- Add 150 µL of 50% glycerol to 350 µL of cells
- Place in -80C freezer

### Transformation Protocol

- With a pipette tip, punch a hole through the foil cover of the DNA plate
- Add 10 µL of DI water
- Thaw competent cells on ice
- Add 1-2 µL of resuspend DNA and 50 µL of thawed competent cells to labeled tubes
- Incubate the cells on ice for 30 minutes
- Heat shock the cells at 42 degrees Celsius for 45 sec
- Incubate the cells on ice for 2 minutes
- Under flame, add 450 µL SOC broth
- Incubate at 37 degrees Celsius for 1 hour while rotating or shaking at 300rpm
- Spread cells on appropriate antibiotic LB plates (usually 100 µL)
- Incubate at 37 degrees Celsius for 18-24 hours
- Take a colony, put in 3 mL of LB + appropriate antibiotic
- Use resulting culture to miniprep DNA and make your own glycerol stock

### Point mutation Protocol

1) Create reaction mixture

        5 ul 10x buffer

        10-100 ng DNA

        1 ul of foward primer

        1 ul of reverse primer

        1 ul of dNTP'S

        1.5 ul of Quik Solution reagent

        Bring to 50 ul with NF-H20

        \*Then add 1ul Quik Change Lightning Enzyme

2)Run thermo-cycler (program--mutate)

        1 cycle: @ 95C 2 minutes

        18 cycles:

                a) 95C x 20 seconds

                b) 60C x 10 seconds

                c) 68C x 30 seconds/kb per plasmid length

        1 cycle: 68C 5 minutes

3) Then add 2 ul of DpnI enzyme directly to each amplification reaction

4) Pipette up & down several times

5) Incubate @ 37C x 5 minutes to digest the parent DNA (cuts methylated dna)

6)Then transform.

## Calendar

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

7/5/2013

Transformation

[GFP] Kit plate 5. Well 14k. Backbone pSB1A2. Resistance A. i5-5-1

[LacI] Kit plate 5. Well 20. Backbone pSB1A2. Resistance A. i5-5-2

7/6/2013

Colony Picking

I5-5-1 ?  2 colonies ? picked 2 ? i5-5-3

                                        ? i5-5-4

I5-5-2 ? 0 colonies

7/7/2013

Mini Prep

I5-5-3: 67.4 ng/ul        [GFP]

I5-5-4: 98.7 ng/ul        [GFP]

I5-5-5: 408.7 ng/ul        [+ confiol]

Test Digestion

|  |  |  |
| --- | --- | --- |
|  | I5-5-31 | I5-5-42 |
| DNA | 4 | 2 |
| BSA | 1 | 1 |
| Buffer 4 | 1 | 1 |
| EcoRI | 1 | 1 |
| Pst | 1 | 1 |
| H2O | 2 | 4 |
|  | 10 | 10 |

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