Team:ULaVerne Collab/Experiments


Gibson Assembly

1.Set up the following reaction on ice:

2-3 Fragment Assembly4-6 Fragment AssemblyPositive Control**
Total Amount of Fragments0.02–0.5 pmols* X μl0.2–1 pmols* X μl10 μl
Gibson Assembly Master Mix (2X)10 μl10 μl10 μl
Deionized H2O10-X μl10-X μl0
Total Volume20 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.

Scanning Electron Microscopy: This is part of our future directions.