Week 6 Lab: Gibson Assembly

Louisa, Yutong, Jasmine and I worked together to complete this lab.

Day 1: PCR and DNA Purification

PCR

First, we performed PCR to amplify the backbone and color DNA fragments. We prepared the PCR reactions according to the tables below, and ran the PCR program on the thermocycler.

Ice bucket
Phusion HF PCR Master Mix
Primers (5 uM stock)
UltraPure Water
PCR tubes
Thermocycler
P20 pipette and 10uL tips
P200 pipette and 200uL tips

Backbone DNA Fragment (Primers: Backbone Fwd and Backbone Rev)

Stock Conc.	Desired Conc.	Volume (uL)
Template mUAV Plasmid	38.5	20	0.8
Backbone Forward Primer	5 uM	0.5 uM	2.5
Backbone Reverse Primer	5 uM	0.5 uM	2.5
Phusion HF PCR Mix	2X	1x	12.5
Nuclease-free water			6.8
Total Volume			25.0

Color DNA Fragment (Primers: Color Fwd and Color Rev)

Stock Conc.	Desired Conc.	Volume (uL)
Template mUAV Plasmid	38.5	20	0.8
Color Forward Primer	5 uM	0.5 uM	2.5
Color Reverse Primer	5 uM	0.5 uM	2.5
Phusion HF PCR Mix	2X	1x	12.5
Nuclease-free water			6.8
Total Volume			25.0

Below are the photos of the PCR tubes in the thermocycler:

After the PCR program was completed, we ran the E-gel. We observed clear bands at the expected sizes for both the backbone and color fragments, indicating that the PCR was successful.

DNA Purification:

PCR products
Zymo DNA Clean & Concentrator
UltraPure Water
1.5ml microcentrifuge tubes
50ml Falcon tube for liquid waste
Centrifuge (Set to 13,000 rpm, or roughly 17,900 x g)
Nanodrop/Qbit
P200 pipette with 200uL tips
P20 pipette with 20uL tips

Day 2: Gibson Assembly and Transformation

Gibson Assembly

Backbone fragment purified, Color fragment(s) purified
Gibson Assembly Master Mix
PCR tubes
UltraPure Water
Thermal Cycler
P20 pipette and 10uL tips
Ice Bucket

Reagent	Stock Conc. (ng/uL)	Desired Conc (ng/uL)	Volume (uL)
Backbone Fragment	50	25	0.5
Color fragment (Single)	50	50	1.0
Gibson Assembly Mix	2X	1X	5
Nuclease-free water			3.5
Total Volume			10

Transformation

Result

After 72 hours of incubation: