Gibson Reaction

Set-up:
If this is your first time doing a Gibson Reaction, refer here for more information on primer design and the basics.

Before doing Gibson Reaction, make sure you have completed the first steps of successful Cloning.

For making Gibson Master Mix and ISO Buffer please refer to here.

You'll Need:

ICE

DI H20: on benchtop

Gibson MM (in -80)

All Necessary Fragments and Vector

XL10 Cells from (-80)

Plates with correct antibiotic from the 4C (inside the MOM Lab)

Calculations:
Our Gibson assembly master mix is 1.3X

Use 3.75uL in a 5uL total volume (or 7.5uL in a 10uL total volume)

First, you will need to do calculations for your Gibson Reaction. See the Gibson Calculations Page for a step by step break down on how to calculate how much of each fragment and vector to add to your reaction. (This should be done in a PCR sized tube)

In our example on the Gibson Calculations Page, we came up with:

7.5uL of Gibson Master Mix

0.75uL of Vector

0.5uL Fragment A

0.5uL Fragment B

Water to bring the final volume to 10uL (0.75uL of water).

Protocol:

 * 1) Incubate at 50 degrees C for 15-60 min in the PCR machine.
 * 2) During the incubation, take plates (with the correct drug: i.e. if your vector has Kanamycin resistance, you'll need plates with KanMX) and put at 37 degrees C.
 * 3) Transform into XL10Gold Competent Cells using the Transformation Protocol
 * 4) See https://www.neb.com/~/media/Catalog/All-Products/0AA961B294E444AFBEDD5C4A904C76E6/Datacards%20or%20Manuals/ManualE2611.pdf

Transformation Protocol:
cells'''
 * 1) Add half* of your Gibson reaction to 50uL of cells. *'''don’t exceed 5uL of Gibson reaction in 50ul of
 * 1) Incubate on Ice for 20 min.
 * 2) While incubating, fill the 42 heat-block with water
 * 3) After the 20 minute incubation on ice, heat shock at 42degC for 50 sec
 * 4) Place the tube directly back on ice
 * 5) Add 500uL SOC medium and incu-shake at 37degC for 2 hrs.
 * 6) Plate 50uL and incubate plates at 37degC incubator overnight.
 * 7) In the morning, remove your plates and store in the 4C.