Team:NTU-Singapore/Notebook/Protocols

Our Protocols

Plasmid resuspension from IGEM paper spots

(Estimated time: 25 min + 5 min for every part if you use scalpel/tweezers or + 15 min for every part if you use punch tool)

Materials needed :

• Pre-warmed at 42°C TE
• Desired spot location information
• punch tool
• ddH20
• 99% ethanol
• 0.5 ml tubes

Protocol

1. Put 10 µL of pre-warmed TE into a 0.5 ml tube.
2. Cut paper spots using punch tool, following the instructions provided with the IGEM kit.
3. Put the cut paper into the 0.5 ml tube.
4. Clean punch tool with water and ethanol every time after cutting a spot; be careful to dry the punch tool.
5. Incubate at 42°C for 20 min.
6. Vortex and spin down.
7. Pipttee 4-6 µl of DNA resuspension for transformation

Plasmid resuspension from Well

(Estimated time: 25 min)

Materials Needed

• DI water

Protocol

1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure the plate is oriented properly. It is NOT recommended to remove the foil cover, as it could lead to cross contamination between the wells.
2. Add 15uL of diH2O (deionized water)
3. Take 2uL DNA to transform your desired competent cells

Side note:

1. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 18 hours.
2. Use the resulting culture to miniprep the DNA AND make your own glycerol stock

BioBrick digestion with restriction enzymes

(estimated time: 3 hours)

Materials needed: Double Digestion mixture composition (total volume: 50 µL)

• DNA 1mg (usually 4~5 µL)
• Enzyme-1 1 µL
• Enzyme-2 1 µL
• NEBBuffer-2 5 µL (1/10 of total volume, check double digestion table for which buffer to use)
• BSA 0.5 µL (1/10 of buffer volume)
• Mili-Q water 37.5 µL

Protocol

1. Measure plasmid concentration by Nano-drop
2. Pipette apropraite amount of vector (usually 4~5 µl for 1 µg)
3. Take out NEBuffer and BSA to thaw (usually stored at -20 deg C), vortex for well mix
4. Adding the following components sequentially into 200 µL PCR tube, following the order: Mili-Q water → Enzyme Buffer → BSA (optional) → DNA → Enzyme
5. Pulse spin the tube and incubate at 37 deg C for 2 hours (2.5hrs)
6. Do MiniElute PCR purification for all the tubes to remove enzymes and buffers
7. Load the 10 µL plasmid with 2 µL loading dye for gel running

Notes

• check 37 deg C incubator is on, take out buffer, BSA, to thaw and vortex before adding, when adding buffer, enzymes, DNA (not BSA), putting the whole pipette tip into the solution, and then pipette up and down. For adding BSA, just push strongly and fast all the way down.

LB - Agar Plate Preparation Protocol

(Estimated time: 3 hours + 1 hour pouring process)

Materials needed

• LB broth
• Agar
• Autoclave bottle
• Petri dishes

Protocol

1. Weigh 25g of LB-Agar powder mix (or 15g LB powder + 10g agar) per liter of media desired. One liter makes 40-50 plates
2. Select an appropriate bottle and dissolve LB-Agar in autoclaved Mili-Q water. Add a stir bar and use a magnetic stirrer to speed dissolve process
3. Cover the bottle with aluminum foil, and secure the foil with autoclave tape. The foil should be somewhat loose (to avoid building pressure in the bottle while sterilizing and blowing the foil off).
4. Load the bottle into the autoclave, and sterilize it
5. Once the autoclave finishes venting (which can take twice as long as the sterilization proper), Unload the hot bottle using the insulated oven gloves
6. Allow the media to cool until it can be handled without the oven mits.
7. Once media is cool, add appropriate amount of ampicilin stock (stock 50mg/ml, final 100ug/ml, To achieve final concentrations, add 2mL of stock per 1L of media), use the magnetic stirrer (added before autoclave) to mix.
8. Pour directly from the bottle into sterile petri plates. Use a inoculation loop to snuff out bubbles that form during pouring. Bubbles can allow cells to access nutrients without being exposed to the plate's antibiotic, and should be blown out immediately before the gel can set.
9. Allow the plates to stand right side up until the gel sets. Plates should be stored upside down to keep condensation from falling on the media. Store petri plates in the plastic bags they ship in, in the 4°C fridge.

Transformation

(Estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)

Materials needed

• LB agar plates with proper antibiotic added (Ampicilin)
• Thawed Invitrogen TOP10 cells (every tube contains 50 µl of cell suspension)
• Resuspended DNA
• SOC medium

Protocol

1. Put 4-6 µl of DNA resuspension into TOP10 tube.
2. Incubate on ice for 30 min.
3. Heat shock: 42°C for 1 min.
4. Put transformed TOP10 tube on ice for 2mins and then add 200 µl SOC medium into the tube.
5. Incubate for 1 hour at 37°C, 225 rpm.
6. Plate 200 µl of solution on a proper agar plate (with Ampicilin resistence).
7. Incubate overnight at 37°C.

Inoculation for overnight growth

(Estimated time: 10 min + 12-16 hours overnight incubation)

Materials needed

• 50mL Falcon tube.
• 5 mL LB medium
• Suitable Antibiotic
• Single colonies on a plate

Protocol

1. Pipet 5uL 1000X antibiotic into culture tube
2. Add 5mL LB medium with Ampicilin resistance
3. Select a single colony using a sterile inoculation loop
4. Place inoculation loop in culture tube and stir
5. Remove inoculation loop and place culture tube in incubator at 37°C overnight shaking vigorously (250 rpm)

Agarose Gel Preparation and Electrophoresis

(Estimated time: 2 hours)

Materials needed

• 1X TAE
• 125 mL flask
• Agarose
• Gel Pouring Tray
• Tape
• Gel rig

Protocol

1. Measure out 40mL of 1X TAE buffer
2. Transfer buffer to 125 mL flask
3. Weigh out 0.4g agarose to make a 1% gel
4. Transfer agarose to 125mL flask
5. Melt agarose in microwave for 30 seconds,
6. Take out the flask and shake it gently
7. Again,put the flask in the microwave for 30 seconds
8. Assemble the gel pouring apparatus by inserting gate into slots.
9. Allow gel to cool until flask can be handled comfortably
10. Add the stain (4 ul for 40 ml TAE) and shake gentely to mix.
11. Place comb in the gel rig
12. Pour agarose into gel tray
13. Allow to solidify. While the gel is solidifying prepare the samples. Mix 10 µl DNA sample with 2 µl 6X loading dye for each slot.
14. Pour 1X TAE over gel so that gel is covered by a 3-5mm buffer
15. Load samples into lane (Don't forget to load 5 µl 1kb+ ladder into one of the lanes)
16. Hook electrodes to gel apparatus
17. Run the apparatus at 120V for 45 minutes
18. Visualize the gel and record the results

Preparation of Top 10 chemical competent cells

(Estimated time: 2 hours)

Materials needed

• Top10 Ecoli cell
• 50 mL centrifuge tubes
• 1.5 mL small tubes
• LB agar plate
• LB medium
• Chilled 0.1 M ice-cold MgCl2
• Chilled 0.1 M ice-cold CaCl2
• Chilled 0.1 M CaCl2 / 15 %glycerol

Protocol

1. Steak a loopful of Top10 Escherichia coli onto a fresh LB agar plate (without selective antibiotics) and incubated at 37 oC overnight.
2. Inoculate a single isolated colony of the Top10 E. coli into 5 ml of LB broth (without selective antibiotics) and incubated with shaking at 250 rpm overnight in a 37oC shaking incubator overnight.
3. Scale up the bacteria culture 100 times into 100 ml of fresh LB and grow to OD 600nm of 0.3 to 0.4 (2-3 hours). The current culture is in the exponential phase
4. Aliquot the above culture into two 50 ml pre-chilled centrifuge tubes
5. Incubate the two 50 ml tubes on ice for 10 min.
6. Centrifuge the two tubes of Top10 cells for 5 min at 5,000 rpm at 4 °C.
7. Decant the supernatant and re-suspend cells pellet in 30 ml of 0.1 M ice-cold MgCl2 thoroughly for each tube.
8. Centrifuge the mixture in the two tubes for 5 min at 5,000 rpm in 4 °C.
9. Decant the supernatant and re-suspend cells pellet in 20 ml of 0.1 M ice-cold CaCl2 for each tube
10. Incubate on ice for 30 min.
11. Centrifuge the mixture in the two tubes for 5 min at 5,000 rpm in 4 °C.
12. Decant the supernatant and re-suspend cells pellet in 1.5 ml of ice-cold 0.1 M CaCl2 / 15 %glycerol for each tube
13. Aliquot the cell suspension into 60 X 1.5 mL tubes each with 50 µl cell suspension
14. Store in - 80 °C deep freezer before use.

DNA concentration Measurement using NanoDrop

(Estimated time: 10~20 mins)

Materials needed

• DNA suspension in EB
• NanoDrop ND-1000 Spectrophotometer

Protocol

1. Start up software by clicking on shortcut
2. Chose measurement (Nucleic acid for DNA and RNA samples)
3. Clean pedestals with tissue with MilliQ water
4. The software asks for a testmeasurement with MilliQwater: Add 1.8 μl of MilliQ water to front pedestal and click “OK”
5. Add 1.8 μl of a Blank (DNA: EB) to pedestal and click “Blank”.
6. Add 1.8 μl of sample, click “Measure”, note results and type in a sample name in the software window
7. Several different values can be read:
8. DNA or RNA conc. in ng/microliter at 260 nm
9. Protein conc. at 230 nm
10. Quality at 260:280 (should be around 1.9)
11. Clean pedestals with tissue before measuring next sample
12. Close software by clicking “Exit” (twice)
13. Retrieve data by clicking on shortcut to “nanodrop data”

SDS Page for protein

(Estimated time: 2~3hrs)

Materials needed

1. SDS page kit (invitrogen)
2. Overnight cell culture

Protocol

1. Inoculate interested colony in LB medium at 37°C overnight (250 rpm)
2. Dilute the above cell culture to 100 times in flask with LB (0.5mL in 50mL LB)
3. Incubate the flask until the culture OD ~= 0.8
4. Divide the above cell culture into two flask each with ~25mL
5. One flask is for control while the other one for experiment
6. Incubate until OD=1.6
7. Pipette 1mL cell culture from the above two flask and centrifuge at 12000rpm for 10 mins
8. Re-suspend the cell in 150 µL TE buffer
9. Pipette 15 µL cell sample into two fresh 1.5mL tubes
10. Add 15 µL sample buffer into the above two tubes
11. Let the tubes stands in boiling water for 10 mins
12. Take the gel out of fridge for it to thaw
13. Spin down the vapor and Load 15 µL sample into each slot
14. Gel run at 200V for 30mins
15. Remove the cover and extract out the gel
16. Wash and stain for 1~2hrs
17. Wash and de-stain for overnight
18. check gel band by densitometer

Notes

• Sample buffer lyses the cell and bind to the protein
• After staining, the entire gel show blue color, the de-stain procedure is to remove the straining, however, the site where protein resides will remain blue since stain bind to the protein