Team:ULaVerne Collab/Experiments
Experiments
Gibson Assembly
1.Set up the following reaction on ice:
| 2-3 Fragment Assembly | 4-6 Fragment Assembly | Positive Control** | |
|---|---|---|---|
| Total Amount of Fragments | 0.02–0.5 pmols* X μl | 0.2–1 pmols* X μl | 10 μl |
| Gibson Assembly Master Mix (2X) | 10 μl | 10 μl | 10 μl |
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μl*** | 20 μl*** | 20 μl |
* Optimized cloning efficiency is 50–100 ng of vectors with 2–3 fold of excess inserts. Use 5 times more of inserts if size is less than 200 bps. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%. ** Control reagents are provided for 5 experiments. *** If greater numbers of fragments are assembled, additional Gibson Assembly Master Mix may be required.
2.Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at -20°C for subsequent transformation.
Gel Electrophoresis
500 mL of TAE Buffer
1. Add 10 mL of TAE buffer
2. 490 mL of D.I. water
1% gel
1. Add 0.5 g of Agarose
2. Add 50 mL of TAE
3. Microwave for 1 min and 45 sec
4. Let it sit until room temperature
5. Add 5 uL of SYBR Safe DNA Gel Stain
6. Swirl Solution
7. Pour gel into cast
8. Let it sit for 20 minutes.
Loading of gel
1. Well 1: 6uL of the 1Kb ladder or 5uL of the APEX ladder
2. Other wells: 6 uL of your DNA (5 parts DNA, 1 part Dye)
SDS PAGE: Protein
8% Gel - Resolving
Add 4ml of 30% Acrylamide/bis 0.5M
Add7.03 ml of Deionized water
Add 3.75 ml( pH 8.8)
Add 150 uL 10% SDS
Add 75 uL 10% APS
Add 7.5 uL of TEMED
Approximately 15ml
5% Stacking
Add 1.3 ml of 30% Acrylamide/bis 0.5M
Add 5.5 ml of Deionized water
Add 1ml of Tris (use pH 6.8)
Add 80 ul of 10% SDS
Add 80ul of 10% APS
Add 8ul of TEMED
Plastic Weighing: We weighed the plastic particles with the laboratory weighing scale before and after inoculation with transformed E. coli+Media.