Transformation Protocol
Goal: To transform parts from the registry into competent cells
Materials:
Procedure:
Expected Data:
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:
- Prepare lab space
- Wipe counter with ethanol
- Light flame
- 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
- Warm up Water Bath to 42°C
- If water bath is too warm, remove some water and add cold water
- 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
- 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
- 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
- Add 1μL of the RFP DNA to the RFP control tube (The RFP control is used as a standard for transformation efficiency measurements)
- Incubate tubes on ice for 45 minutes
- 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
- Put cells back on ice for 5 minutes
- Add 200μL of SOC media to each tube
- Incubate cells for 2 hours at 37°C and at 250 rpm (to aerate cells and evenly distribute cells within nutrients)
- 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)
- Plating the Cells
- 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
- Plate growth images
- Images of RFP fluorescence