Team:UIUC Illinois/Experiments

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A. Using a Lab Notebook

1. Always include the date and your initials or signature.
2. Procedures from previous labs do not have to be rewritten, but should be clearly referenced.
3. Always Include:
   • Any and all calculations
   • Number of cells seeded
   • Components of solutions
   • Type of cells
   • Type of media
   • Brand names of solutions used (Invitrogen, Bio-Rad, Promega, etc.)
   • Observations such as confluency, color of media, etc.
4. Always write directly in the notebook, rather than writing it on a separate sheet and copying it later.
5. Do no leave large open spaces. If you do, cross these out.
6. If you cross out text, make sure it is still legible underneath.
7. Number all pages in the notebook, and do not tear pages out.
8. Write all entries in ink.
9. Use original documentation when possible (e.g. machine printout rather than copying data by hand.)
10. Be thorough! Somebody should be able to repeat your experiments by looking at the lab notebook.

B. Making LB

1. Add 20 g of LB powder to 1 L of water and mix.
2. Autoclave on a liquid cycle.
3. If adding antibiotics, wait until the LB has cooled enough to comfortably touch the bottle. Otherwise, the heat will start to break them down.

C. Pouring plates

1. Add 20 g of LB powder and 14 g of agar (NOT agarose) to 1 L of water and mix.
2. Autoclave on a liquid cycle
3. Wait until the LB agar has cooled enough to comfortably touch the bottle.
4. If desired, add antibiotics now.
5. Pour 10 - 20 mL of LB agar into each plate. This can be done by eye, but using a pipet-aid will give more consistent results
6. If any bubbles formed on the surface of your plates, quickly pop them using a pipet tip.
7. Allow the plates to solidify.
8. Wrap plates in a bag, sleeve, or Parafilm.
9. Store upside down at 4 degrees.

D. Transformation

1. Take competent cells out of freezer and thaw on ice for 20-30 minutes. Take only as many as you need; they lose competency if re-frozen.
2. Take agar plates out of storage and allow them to warm up to room temperature.
3. Add 10 pg to 100 ng of DNA (usually about 1-5 uL depending on concentration) to 20 - 50 uL of competent cells.
4. Mix tubes by gently flicking.
5. Place tubes back on ice for 20-30 minutes.
6. Heat shock transformation by placing it in 42 degree C water bath for 45 seconds.
Note: Some strains of e coli may require shorter or longer heat shock times. Always check the strain before transforming!
7. Set tubes back on ice for 2 minutes.
8. Add 250 - 500 uL of LB and grow in the 37 shaking incubator for 45 minutes. If you are plating on ampicillin, this step can be skipped.
9. Plate 50 uL of cells on each plate using beads or cell spreader.
10. Grow overnight at 37 degrees C.

E. Making electrophoresis gels

1. Add 1 g of agarose to 100 mL of 10x TAE buffer.
2. Heat in the microwave for about 1 minute and 45 seconds, until the solution has come to a boil.
3. Allow the liquid to cool for 5 minutes.
4. Add 10 uL of Midori Green and swirl gently to mix.
5. Pour 30 - 50 mL into gel tray with comb. Use as little gel as possible if you plan to extract and purify plasmids from it later.
6. Let sit about 30 minutes to cast.
7. Note: this makes a 1% gel, which works for most purposes. However, a lower percentage, such as 0.7% gel, is best for gel extractions, and as high as 2% agarose can be used when resolving large bands of DNA.

F. Gel Electrophoresis

1. Cast an agarose gel and set it in the electrophoresis box.
2. Add 1 uL of 6x loading dye for every 5 uL of sample.
3. Load a DNA ladder into the first and last wells of the gel.
4. Load one sample into each remaining lane of the gel, being careful not to puncture the well or let the sample overflow.
5. Run gel at 120 V for approximately 25 minutes.
6. Image gel using the Ethidium Bromide protocol on the gel imager (we use Midori Green, but the EthBr protocol also works).

G. Plasmid Purification

1. Grow up a colony overnight in 5 mL of LB in the 37 degree C shaker.
2. Follow the protocol included with the DNA mini-prep kit you are using. We used the Omega Bio-Tek E.Z.N.A. Plasmid DNA Mini Kit.

H. Double Digestion (dephos, dont automatically assume cutsmart, dpn1, consider glycerol)

1. Use a Double Digest finder, such as that on the NEB website, to find the optimal buffer for your reaction.
2. Add the directed amount of your chosen buffer, and BSA if necessary
3. Add 0.75 uL of each enzyme.
4. Add DNA to be digested.
5. Bring up the total volume to 7.75 uL

I. PCR for Amplification.

1. For each PCR, we followed the protocol included with the Q5 High-Fidelity Polymerase from NEB. Each PCR varied slightly depending on what was being amplified; amounts of each component are included in the lab notebook each time a PCR was performed.

J. Colony PCR

1. Choose approximately 8 colonies to check for the correct sequence.
2. Lightly touch each colony with a pipet tip and dilute in 50 uL LB.
3. For each reaction, use:
   • 0.25 uL of forward primer
   • 0.25 uL of reverse primer
   • 5 uL of Green GoTaq
   • 1 uL of template (your dilution from step 2)
   • 3.5 uL H20
4. Run a thermocycler program according to the instructions included with your polymerase.

K. Making Competent Cells (OD and growth phase) (test efficiency w/ puc19)

1. Prepare the following:
   • LB
   • 0.1 M CaCl2 (keep cold)
   • 0.1 M CaCl2‐15% glycerol (keep cold)
   • 1.5 ml eppendorf tubes (keep cold)
2. Grow 50 uL of old comp cells in a 5 ml preculture overnight in LB
3. Make a 1% inoculation to 50 ml LB (in 250 ml flask)
4. Place in shaker at 37°C until OD 0.5 (takes about 1.5 hours)
5. Set centrifuge to 4 degrees C.
6. Transfer 50 ml culture to 50 ml falcon tube
7. Centrifuge for 10 min at 3000 rpm at 4°C
8. Remove supernatant as much as possible and resuspend cells in 5 ml 0.1 M
CaCl2 using pipette tips
9. Rest the tube for 30 min on ice
10. Repeat 7-9 two more times, and then proceed to 11
11. Centrifuge with same settings as 7, and then completely remove supernatant
12. Resuspend cells in 2.5 ml of 0.1 M CaCl2‐15% glycerol
13. Aliquot 50 ul to sterile 1.5 ml tubes and keep in ‐80°C

L. 3A Assembly (We use the iGEM Registry protocol)

1. Create the Enzyme Master mix for the plasmid backbone:
   • 5 uL NEB Buffer 2
   • 0.5 uL BSA
   • 0.5 uL EcoRI-HF
   • 0.5 uL PstI
   • 0.5 DpnI
   • 18 uL dH20
2. Create the Enzyme Master Mix for Part A:
   • 5 uL NEB Buffer 2
   • 0.5 uL BSA
   • 0.5 uL Eco-RF
   • 0.5 uL SpeI
   • 18.5 uL dH20
3. Create the Enzyme Master Mix for Part B:
   • 5 uL NEB Bugger
   • 0.5 uL BSA
   • 0.5 uL XbaI
   • 0.5 uL Pst1
   • 18 uL dH20
4. Add 4 uL of linearized plasmid backbone to 4 uL of Enzyme Master Mix for Backbone
5. Add 100 ng (4 uL) of Part A to 4 uL of Enzyme Master Mix for Part A.
6. Add 100 ng (4 uL) of Part B to 4 uL of Enzyme Master Mix for Part B.
7. Digest all three reactions (from steps 4-6) at 37 degrees C for 30 minutes
8. Heat kill the enzymes at 80 degrees C for 20 minutes
9. Add 2 uL of digested Plasmid Backbone
10. Add an equimolar amount of digested Part A.
11. Add an equimolar amount of digesed part B.
12. Add 1 uL of T4 DNA ligase buffer.
13. Add 0.5 uL of T4 DNA ligase.
14. Add enough H20 to bring reaction total to 10 uL.
15. Ligate at 16 degrees C for 30 minutes.
16. Heat kill at 80 degrees C for 20 minutes.
17. Transform with 1-2 uL of product.

M. Gibson Assembly

1. Use the protocol included in the kit you purchase. (We used an NEBuilder kit.)