Once our Gene fragments had been ordered, it was necessary to assemble them into our destination plasmid, pET28a.
Step 1: Design fragments (g blocks) and order the necessary primers to open destination plasmid and prep plasmid for Gibson Assembly.
Step 2: Amplify Plasmid and purify
Step 3: Perform Gibson Assembly Reaction
Note: All components should be kept on ice
| Component | Amount |
|---|---|
| Fragments (G-Blocks) | 1 ul of each fragment |
| Plasmid Vector | 2 uL of vector |
| 2x Gibson Assembly Master Mix | 10ul |
| Mili-Q Water | Adjust to final volume of 20ul |
| Total | 20ul |
Step 4: Incubate in thermocycler for 50ºC, for one hour
Step 5: Transform into competent E.Coli cells
Step 6: Run colony PCR in order verify the assembly was successful
The purpose of PCR was to amplify sections of our DNA of interest. We used PCR to check if our Gibson assembly was successful by using special primers that flanked the insert region, to validate successful assembly of our target sequence.
Step 1: Primer Reconstitution
Spin lyophilized primer vials for 3 minutes at 6800g
Resuspend primers to a final concentration of 100 nM
Prepare 10 nM stock of both forward and reverse primers by diluting with DI water
Step 2: Prepare 50 µL PCR reaction mix
| Component | Amount | Final Concentration |
|---|---|---|
| Q5 2X master Mix | 25 µL | 1X |
| Forward Primer | 2.5 µL | 0.5 µM |
| Reverse Primer | 2.5 µL | 0.5 µM |
| Template DNA | Variable | ~50-100ng |
| DI water | To 50 µL | - - |
Step 3: Thermocycling Protocol for Amplification of Desired Segment
| Temperature | Time (s) | |
|---|---|---|
| Denaturation (1): | 94°C | 30 |
| Denaturation (2): | 94°C | 5 |
| Anneal*: | 60-72°C | 25 |
| Extend (1): | 72°C | 30 seconds/ Kb |
| Cycle Number: | 28 | |
| Extend (2): | 72°C | 5 minutes |
| Hold: | 4°C | Indefinitely |
*Will vary depending on primer
Step 4: Purify using Monarch PCR purification kit: elute 30 µL into a 1.5 mL tube
Step 5: Nanodrop purified product by adding 1uL of the sample to check for concentration (ng/uL)
Note: Blank nanodrop with elution buffer to calibrate
To test if our Gibson assembly reactions were successful, we picked colonies from the transformation plates and ran a colony PCR reaction. Any successful colonies were then sent for sequencing. Typically, we ran 8-10 colonies.
Step 1: Pick large, well isolated single colonies and use to inoculate 100 uL’s of nuclease free water.
Step 2: Prepare 50 µL reaction mix.
| Components | Amounts |
|---|---|
| One Taq 2x Reaction Buffer | 25 uL |
| Forward Primer | 1 uL |
| Reverse Primer | 1 uL |
| Template DNA (individual colonies dissolved in H2O) | 10 uL |
| DI Water | 13 uL |
| Total | 50 uL |
Step 3: General thermocycling Protocol for Menthol and Linalool Amplification.
| Temperature | Time (s) | |
|---|---|---|
| Denaturation (1): | 94°C | 30 |
| Denaturation (2): | 94°C | 5 |
| Anneal: | 46°C* | 25 |
| Extend (1): | 68°C | One minute / Kb |
| Cycle Number: | 30 | |
| Extend (2): | 68°C | 300 |
| Hold: | 4°C | Indefinitely |
*Will vary by primer
Step 4: The entire colony PCR reaction is then run on a DNA gel to determine if the appropriate DNA was inserted.