Goal:

To transform parts from the registry into competent cells Materials:

• Parts from Kit Plates
• Plates with Antibiotic Resistance (Must be warmed up in 37°C room)
  • 2 plates per part (specific antibiotic type depends on part resistance)
  • 1 plate for negative control (for each type of antibiotic used)
• 1 Ampicillin plate (for RFP control)
• 1 LB Plate (for positive control)
• 10 pg/μL RFP control
• Eppendorf Tubes
  • 2 tubes for controls
  • 1 tube per part
• PCR Tubes
  • 1 tube per part
• BL21 Competent Cells
  • 50μL for controls
  • 25μL per part
• SOC Media
  • 400μL for controls
  • 200μL per part
• Ice
• 42°C Water Bath
• Glass Beads
• 37°C Incubator
• Pipettes and Tips (Filtered Tips if possible)
• Deionized Water

Procedure:

1. Prepare lab space
   • Wipe counter with ethanol
   • Light flame
2. Resuspend parts from the kit plates and transfer them to labeled PCR tubes
   • Mark part location on kit plate with a Sharpie marker
   • Use pipette tip to puncture kit plate and move around to clear all foil from opening
   • Add 10μL of DI Water to Kit Plate well and pipette up and down until orange liquid is present in pipette tip (The DNA is freeze-dried and needs to be resuspended in water)
   • Put the 10μL of DNA suspension into a labeled PCR tube
   • Repeat for each necessary part
3. Warm up Water Bath to 42°C
   • If water bath is too warm, remove some water and add cold water
4. Thaw competent cells and RFP DNA on ice
   • Fill ice bucket with ice from the autoclave room
   • Bring the ice bucket to the -80°C freezer and immediately put competent cells in ice when removing from freezer
   • Retrieve RFP DNA from -20°C freezer and immediately put in ice
5. Add 25μL of competent cells to each of the eppendorf tubes. Label each.
   • Keep cells in ice as much as possible
   • Mark top of cell container with Sharpie (to mark that they have been used)
   • Return cells to -80°C freezer as soon as possible
   • Label each eppendorf tube with the part name, RFP Control, or Control
6. Add 4μL of DNA to respective eppendorf tube (except the RFP control and Control tubes)
   • iMake sure that the DNA and cells are mixed well
   • Store leftover DNA (in PCR tube) in -20°C Freezer
7. Add 1μL of the RFP DNA to the RFP control tube (The RFP control is used as a standard for transformation efficiency measurements)
8. Incubate tubes on ice for 45 minutes
9. Heat shock cells at 42°C for 60 seconds (to open the cell walls so that the DNA can enter the cell)
   • Insert eppendorf tubes into Styrofoam holders (so they float in the water bath)
   • Tubes must remain in ice until insertion into water bath then swiftly move tubes to water bath
   • Time very precisely and then immediately place tubes back into ice after 60 seconds
   • After about a minute, move tubes around in ice because the nearby ice has probably melted
10. Put cells back on ice for 5 minutes
11. Add 200μL of SOC media to each tube
12. Incubate cells for 2 hours at 37°C and at 250 rpm (to aerate cells and evenly distribute cells within nutrients)
13. Plate cells
    • Plating the Cells
      • Scatter the appropriate amount of the cell suspension in drops on the plate
      • Pour about 5 glass bead onto the plate
      • Swirl plate to move glass beats around and evenly coat media with liquid suspension of cells
      • Ensure that plates are properly labeled
    • All parts should be plated at 20μL and 100μL on antibiotic plates (Be sure to use correct antibiotic plate depending on part resistance)
    • The RFP Control should be plated on an Ampicillin plate at 100μL
    • Plate 100μL of the cells on antibiotic plate(s) (Negative control)
    • Plate 100μL of cells on LB plate (Positive control)
14. Incubate overnight from 12-18 hours (at 37°C)

Expected Data:

• Cell growth observed on the Positive Control
  • Ensures that cells are growing properly
• No Cell growth observed on the Negative Control
  • Ensures that the antibiotic is functioning
• Observed growth and fluorescence on the RFP control plate
• Cell growth on the plates with the parts
  • Less growth should be observed on the 20μL plates because fewer cells were placed on the plates to grow originally

Recorded Data:

• Plate growth images
• Images of RFP fluorescence

Conclusions:

If growth was observed on the plates with the transformed parts, no growth on the negative control, and growth on the positive control, then the transformation was successful. Next, the transformed parts must be mini-prepped.

Goal:

To isolate and extract genomic DNA from transformed colonies

Materials:

• Qiagen Miniprep Kit
  • Buffer P1 is kept in the 4°C
• Centrifuge
• Micropipette and Tips
• Eppendorf Tubes
• Qiagen Spin Columns
• Cells in liquid media

Procedure:

• (This only needs to be done once) - Add LyseBlue to Buffer P1 at a ratio of 1:1000)
• Warm up EB Buffer in water bath to 50°C
  • Place EB Buffer in Styrofoam holder to float in water bath
• Pellet 1-5 mL of bacterial overnight culture by centrifugation at >8000 rpm for 3 minutes at room temperature
  • Add 1mL of each liquid cell culture to each eppendorf tube
  • Place eppendorf tubes in centrifuge (balanced)
  • Centrifuge for 3 minutes at 10,000 rpm
  • Pour out liquid into sink (Pellet should still remain at bottom)
  • Continue process, add liquid cell culture to eppendorf tube with pellet until all of the cells have been pelleted
• Resuspend pelleted cells in 250μL Buffer P1 and transfer to a micro-centrifuge tube (Buffer P1 lyses the cells)
  • Can vortex or tap tube on counter to Resuspend the cells (must not be stuck to bottom)
  • Put Buffer P1 back into the 4°C fridge
• Add 250μL Buffer P2 and mix thoroughly by inverting the tube for 3 minutes (Buffer P2 also lyses the cells)
• PROMPTLY add 350μL Buffer NE3 and mix immediately by inverting the tube for 1 minute (This neutralizes the lyse buffers. It is important to do this promptly so that they do not damage the DNA)
  • "Should look like a desiccated coconut and not gloopy”
• Centrifuge for 10 minutes at 13,000 rpm (The cells will collect at the bottom of the eppendorf tube and the supernatant (liquid in the tube) contains the DNA)
• Apply supernatant from step 7 to the Qiagen Spin Column. Centrifuge for 60 seconds and discard flow-through (Separates DNA from liquid in the supernatant)
• Add 750μL Buffer PE and centrifuge for 60 sec and discard flow-through (Buffer PE prevents DNA from dissociating from the column while washing away other contaminants)
• Repeat the previous step
• Centrifuge for 1 minute to remove residual wash buffer
• Transfer to a new microcentrifuge tube
• Add 25μL Buffer EB, let stand 1 minute, centrifuge 1 minute (Buffer EB solubilizes the DNA so that it can flow through the spin column)
• Repeat the previous step
• Measure DNA concentrations with the Nanodrop (bring a blank of the EB Buffer)
• When finished, store DNA in the -20°C freezer

Expected Data:

• DNA concentration readings of 100 – 300 ng/μL are great
• DNA concentration readings of 50 – 100 ng/μL are okay

Recorded Data:

• Measure DNA Concentrations using the Nanodrop
  • Located upstairs
  • Must be trained on the Nanodrop

Conclusions:

DNA from the transformations has now been extracted! Future work includes digestion and ligation

We used a premade Becton, Dickinson and Comapany mix to make our LB Agar

How to make LB liquid plus antibiotics:

• Ampicillin – The frozen stock solutions of ampicillin are at 50mg/ml and 100mg/ml in H2O, and are marked with a red sticker. The final concentration for LB liquid culture is 50ul/ml. To obtain this in 100ml (the amount in each LB bottle), add 100ul stock solution.
• Kanamycin – The frozen stock solutions of kanamycin are at 50mg/ml in H2O, and are marked with green. The final concentration for LB liquid culture for growing plasmids is 50ug/ml, and for cosmids is 20ug/ml. To obtain 50ng/ml in 100ml of LB, add 100ul stock solution, and to obtain 20ug/ml, add 40ul stock solution.
• Tetracycline – The frozen stock solutions of tetracycline are at 15mg/ml in methanol and are marked with black. The final concentration for LB liquid culture is 15ug/ml. To obtain this in 100ml of LB, add 100ul stock solution.
• Chloramphenicol – The frozen stock solutions of chloramphenicol are at 25mg/ml in 100% ethanol and are marked with purple. The final concentration for LB liquid culture is 25mg/ml. To obtain this in 100ml of LB, add 100ul stock solution.

How to make LB plates plus antibiotics:

Follow the recipe card in box for making LB plates, being sure to add the agar. After autoclaving, and when the agar has cooled enough that it’s not too hot to touch (about 1 to 1.5hrs), add antibiotics as follows:
• Ampicillin – add 1ml ampicillin (at 100mg/ml) per liter of agar to obtain a final concentration of 100ug/ml. Mark the plate with a single red line on the side.
• Kanamycin – add 1ml kanamycin stock (at 50mg/ml) per liter of agar to obtain a final concentration of 50ug/ml. Mark the plates with a single green line on the side.
• Tetracycline – add 1ml tetracycline stock (at 15mg/ml) per liter of agar to obtain a final concentration of 15ug/ml. Mark the plates with a single black line on the side.
• Chloramphenicol – add 1ml chloramphenicol stock (at 25mg/ml) per liter of agar to obtain a final concentration of 100ug/ml. Mark the plates with a single purple line on the side.

• 2% w/v bacto-tryptone (20 g)
• 0.5% w/v Yeast extract (5 g)
• 8.56mM NaCl (0.5 g) or 10mM NaCl (0.584 g)
• 2.5mM KCl (0.186 g)
• ddH2O to 1000 mL[4]
• 10mM MgCl2 (0.952 g) or 20mM MgSO4 (2.408 g)[2]
• 20mM glucose (3.603 g)

For maximum effectiveness, SOC media should have its pH adjusted to 7.0 by adding concentrated sodium hydroxide. Autoclave media to ensure sterility

The protocol for creating chemically competent cells is provided by Open Wet Ware and can be found here.

The protocol for 3A Assembly is provided by Open Wet Ware and can be found here.

1. Measure out 5mL of SOB media into each culture tube
2. Add 5uL of the cell liquid culture the respective tubes
3. Do not add anything to the last tube to keep as a blank
4. Place the culture tubes in an incubator for 1 h
5. Record the OD after an hour
6. Check and record the OD for every hour until the OD starts to double, then check every 30 minutes
7. Record all data

With special thanks to the Freiburg iGEM Team for providing this protocol!