Reagents | Volume (µL) | |
---|---|---|
1 | Forward Primer (2.5mM) | 2 |
2 | Reverse Primer (2.5mM) | 2 |
3 | One Taq 2x Master Mix | 12.5 |
4 | MiliQ Water | 7.5 |
5 | Colony Sample | 1 |
TOTAL: | 25 |
Step: | Temperature: (ºC) | Time: (seconds) |
---|---|---|
Denaturing | 94 | 30 |
Annealing | 59 | 45 |
Extension | 68 | 20 |
Repeat steps above 10x while decreasing the annealing temperature 0.5ºC per cycle! | ||
Denaturing | 94 | 30 |
Annealing | 54 | 45 |
Extension | 68 | 20 |
Repeat steps above 20x! | ||
Safety Extension | 68 | 300 (5 min) |
Hold | 4 | ∞ |
Reagents | Volume (µL) | |
---|---|---|
1 | Forward Primer (2.5mM) | 10 |
2 | Reverse Primer (2.5mM) | 10 |
3 | Q5 High-Fidelity 2x Master Mix | 25 |
4 | MiliQ Water | 4 |
5 | pET-28:GFP Template Plasmid | 1 |
TOTAL: | 50 |
Step: | Temperature: (ºC) | Time: (seconds) |
---|---|---|
Denaturing | 98 | 30 |
Annealing | 70 | 30 |
Extension | 72 | 150 (2.5 min) |
Repeat steps above 10x while decreasing the annealing temperature 0.5ºC per cycle! | ||
Denaturing | 98 | 30 |
Annealing | 67 | 30 |
Extension | 72 | 150 (2.5 min) |
Repeat steps above 20x! | ||
Safety Extension | 72 | 120 (2 min) |
Hold | 4 | ∞ |
Reagents | Volume (µL) | |
---|---|---|
1 | BsaI-HFv2 | 0.5 |
2 | T4 DNA Ligase | 1 |
3 | 10x T4 DNA Ligase Buffer | 1.5 |
4 | pET-28 linearized backbone (463.9 ng/µL) | 0.3 |
5 | Diluted GLP-1 R insert (26.26 ng/µL) | 1 |
6 | MiliQ Water | 10.7 |
TOTAL: | 15 |
Step: | Temperature: (ºC) | Time: |
---|---|---|
Restriction & Ligation | 37 | 3 hours |
Linearize remaining plasmid | 50 | 5 minutes |
Inactivation (prevent re-ligation) | 80 | 10 minutes |
Hold | 16 | ∞ |
Reagents | Volume (µL) | |
---|---|---|
1 | Forward Primer (2.5mM) | 2 |
2 | Reverse Primer (2.5mM) | 2 |
3 | One Taq 2x Master Mix | 12.5 |
4 | MiliQ Water | 7.5 |
5 | Colony Sample | 1 |
TOTAL: | 25 |
Step: | Temperature: (ºC) | Time: (seconds) |
---|---|---|
Denaturing | 94 | 30 |
Annealing | 53 | 45 |
Extension | 68 | 40 |
Repeat steps above 10x while decreasing the annealing temperature 0.5ºC per cycle! | ||
Denaturing | 94 | 30 |
Annealing | 48 | 45 |
Extension | 68 | 40 |
Repeat steps above 20x! | ||
Safety Extension | 68 | 300 (5 min) |
Hold | 4 | ∞ |
Reagents | Volume (µL) | |
---|---|---|
1 | BsaI-HFv2 | 0.5 |
2 | T4 DNA Ligase | 1 |
3 | 10x T4 DNA Ligase Buffer | 1.5 |
4 | pET-28 linearized backbone (463.9 ng/µL) | 0.3 |
5 | Diluted GLP-1 R insert (26.26 ng/µL) | 1 |
6 | MiliQ Water | 10.7 |
TOTAL: | 15 |
Step: | Temperature: (ºC) | Time: |
---|---|---|
Restriction & Ligation | 37 | 3 hours |
Linearize remaining plasmid | 50 | 5 minutes |
Inactivation (prevent re-ligation) | 80 | 10 minutes |
Hold | 16 | ∞ |
Materials:
Sterile condition:
Procedure:
We used the protocol from Zyppy Plasmid Miniprep Kit from Zymo Research
We followed the protocol for the ZymoPURE II Plasmid Midiprep Kit from Zymo Research
We used adapted our protocol from NEB’s Golden Gate Assembly Protocol for Using NEB Golden Gate Assembly Mix (E1600)
Materials
Protocol
We followed the protocol for the Frozen-EZ yeast transformation II kit from Zymo Research
Protocol Notes
Yeast colonies were grown to saturation overnight in YPD, then diluted 1:100 in 50 mL of fresh media and grown for 4−6 h to OD600−0.8. Cells were pelleted and washed once with water and twice with 100 mM Lithium Acetate (Sigma). Cells were then mixed by vortexing with 2.4 mL of 50% PEG-3350 (Fisher Scientific), 360 μL of 1 M Lithium Acetate, 250 μL of salmon sperm DNA (Sigma), and 500 μL of water. DNA was added to 100−350 μL of transformation mixture and incubated at 42 °C for 25 min. When selecting for prototrophy, the transformation mixture was pelleted, resuspended in water, and plated directly onto solid agar plates. When selecting for drug resistance, the transformation mixture was pelleted, resuspended in YPD, incubated at 30 °C for 2 h with shaking, pelleted and washed with water, then plated onto solid agar plates. Plasmids designed for chromosomal integration (i.e., containing 5′ and 3′ genome homology regions without a yeast origin of replication) were digested with NotI for 10 min prior to transformation to stimulate homologous recombination. The entire digestion reaction (without DNA cleanup) was included in the transformation in place of plasmid DNA
Protocol
Procedure
Molarity (mM) | Volume of 300 mM (mL) | Volume of PBS (mL) |
---|---|---|
50 | 8.33 | 41.67 |
100 | 16.67 | 33.3 |
150 | 25 | 25 |
200 | 33.3 | 16.67 |
250 | 41.67 | 8.33 |
300 | 50 | 0 |