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Transformation Protocol
Goal: To transform parts from the registry into competent cells
Procedure: 1. Prepare lab space a. Wipe counter with ethanol b. Light flame 2. Resuspend parts from the kit plates and transfer them to labeled PCR tubes a. Mark part location on kit plate with a Sharpie marker b. Use pipette tip to puncture kit plate and move around to clear all foil from opening c. 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) d. Put the 10μL of DNA suspension into a labeled PCR tube e. Repeat for each necessary part 3. Warm up Water Bath to 42°C a. If water bath is too warm, remove some water and add cold water 4. Thaw competent cells and RFP DNA on ice a. Fill ice bucket with ice from the autoclave room b. Bring the ice bucket to the -80°C freezer and immediately put competent cells in ice when removing from freezer c. 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. a. Keep cells in ice as much as possible b. Mark top of cell container with Sharpie (to mark that they have been used) c. Return cells to -80°C freezer as soon as possible d. 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) i. Make sure that the DNA and cells are mixed well b. 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) a. Insert eppendorf tubes into Styrofoam holders (so they float in the water bath) b. Tubes must remain in ice until insertion into water bath then swiftly move tubes to water bath c. Time very precisely and then immediately place tubes back into ice after 60 seconds d. 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 • 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) a. Scatter the appropriate amount of the cell suspension in drops on the plate b. Pour about 5 glass bead onto the plate c. Swirl plate to move glass beats around and evenly coat media with liquid suspension of cells d. Ensure that plates are properly labeled 14. Incubate overnight from 12-18 hours (at 37°C) Expected Data: • Cell growth observed on the Positive Control o Ensures that cells are growing properly • No Cell growth observed on the Negative Control o Ensures that the antibiotic is functioning • Observed growth and fluorescence on the RFP control plate • Cell growth on the plates with the parts o 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.
Materials: • Parts from Kit Plates • Plates with Antibiotic Resistance (Must be warmed up in 37°C room) o 2 plates per part (specific antibiotic type depends on part resistance) o 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 o 2 tubes for controls o 1 tube per part • PCR Tubes o 1 tube per part • BL21 Competent Cells o 50μL for controls o 25μL per part • SOC Media o 400μL for controls o 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 a. Wipe counter with ethanol b. Light flame 2. Resuspend parts from the kit plates and transfer them to labeled PCR tubes a. Mark part location on kit plate with a Sharpie marker b. Use pipette tip to puncture kit plate and move around to clear all foil from opening c. 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) d. Put the 10μL of DNA suspension into a labeled PCR tube e. Repeat for each necessary part 3. Warm up Water Bath to 42°C a. If water bath is too warm, remove some water and add cold water 4. Thaw competent cells and RFP DNA on ice a. Fill ice bucket with ice from the autoclave room b. Bring the ice bucket to the -80°C freezer and immediately put competent cells in ice when removing from freezer c. 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. a. Keep cells in ice as much as possible b. Mark top of cell container with Sharpie (to mark that they have been used) c. Return cells to -80°C freezer as soon as possible d. 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) i. Make sure that the DNA and cells are mixed well b. 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) a. Insert eppendorf tubes into Styrofoam holders (so they float in the water bath) b. Tubes must remain in ice until insertion into water bath then swiftly move tubes to water bath c. Time very precisely and then immediately place tubes back into ice after 60 seconds d. 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 • 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) a. Scatter the appropriate amount of the cell suspension in drops on the plate b. Pour about 5 glass bead onto the plate c. Swirl plate to move glass beats around and evenly coat media with liquid suspension of cells d. Ensure that plates are properly labeled 14. Incubate overnight from 12-18 hours (at 37°C) Expected Data: • Cell growth observed on the Positive Control o Ensures that cells are growing properly • No Cell growth observed on the Negative Control o Ensures that the antibiotic is functioning • Observed growth and fluorescence on the RFP control plate • Cell growth on the plates with the parts o 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.
