Week 6: Gibson Assembly

Day One

Materials:

The following items were used:

PCR tubes
Centrifuge tubes
P200 pipette with 200uL tips
P20 pipette with 20uL tips
Nuclease-free water
Sharpie
Tube holder
invitrogen E-Gel EX: Agarose 1%

Biological material I used included:

Backbone fragment purified
Color fragment(s) purified in Light Pink, Blue, Purple
Backbone Forward Primer
Backbone Reverse Primer
Color Reverse Primer
Template mUAV Plasmid
Phusion HF PCR Mix
DNA Binding Buffer

Machines I used included:

Centrifuge
invitrogen E-Gel PowerSnap
PCR Incubator

Part 1: PCR

First, we created PCR mixtures according to the following table, using Light Pink, Purple and Blue as our color primers of choice. So total, there should be 4 PCR tubes: Backbone, Light Pink, Purple and Blue.

We then ran the PCR reaction with the following settings:

Both of these tables were pulled from the HTGAA lab handout.

Part 2: Gel Diagnostic

After the PCR reaction was complete, we had to run a gel diagnostic to ensure this reaction was completed correctly. The protocol was as follows:

Take 2uL of each mixture and transferit into new PCR tubes (labeled).
Pipette 2uL of mUVA into new tube.
Add 20uL of water to each PCR tube.
Unpack gel electrophoresis cassette
Load into machine
Pipette DNA Ladder into first well
Pipette 20uL of mixture from each NEW PCR tube into the correct wells. In total, there were 6 full wells.
Use the automatic setting for 1%, and wait 10 minutes.

Thankfully, it was successful!

Part 3: DNA Purification and Quantification

Pipette 100uL of DNA Binding Buffer into a centrifuge tube
Add 20uL of PCR product
Mix briefly by vortexing
Transfer 120uL of the mixture into separate columns with a collection tube
Centrifuge for 1 minute
Discard the flowthrough
Add 200 uL of DNA wash buffer to the column
Centrifuge for 1 min
Repeat the last two steps
Transfer the column to new tube
Discard flow through
Add 6uL of nuclease free water to the column matrix
Allow it to sit for 2 min
Centrifuge for 1 min
Store and save

Day Two

Materials

Items Used:

P1000 pipette with 1000uL tips
P20 pipette with 10uL tips
PCR Tubes

Biological Materials Used:

Purified Fragments
Gibson Assembly Master Mix
Nuclease Free Water
LB-Agar plates with Chioramphenicol
SOC Growth Medium
DH5α competent cells

Machines Used:

Thermal Cycler
Shaking Incubator
Waterbath set to 42C

Part 1: Setting Up Gibson Assembly

Set up reaction in proportions according to the table below, for each color fragment
Incubate the reaction at 50 C for 30 minutes in a heat block
Add 100 uL of nuclease-free water to dilute sample

Part 2: Transformation

Transfer 20uL of competent cells to each tube
Transfe purified assembly products into each tube (8 total, 3 Light Pink, 3 Blue, 3 Purple)
Incubate on ice for 30 min
Shock the cells by keeping tubes at 42 C for 45 seconds, immediately after the ice bath
Add 100uL of SOC media to each tube
Allow growth in a shaking incubator for 1 hour
Transfer 100uL from each tube to appropriate plate and use plating beads / plastic spreader as needed
Incubate the plates at 37°C for 72 hours

Part 3: Results

All colonies exhibited an indigo color that’s consistent with wildtype amilCP. The red circle is an interesting occurence of a colorless colony.

What may have happened is that there was not the correct molar ratio of insert to backbone, which may have occurred after purification. This meant that the backbone might have ended up in excess and annealed to each other rather than the insert. This explanation also explains why this would have been consistent across all the different volume groups. Had there been too much insert, there would have been mostly colorness colonies. These colonies survive selection and express a wildtype indigo color.

With regards to the transparent colony, it signals that the backbone reassembled without the color insert and does not have the amilCP CDS.

This colony is evidence that the Gibson Assembly process was occuring, just not as we intended.