Tutorial for NEB Golden Gate
Assembly Tool.
In this video, we will demonstrate
how to use the NEB Golden Gate
Assembly Tool.
As an example, we will build an
assembly from five sample inserts,
or modules, using the PGGa
select destination plasmid
supplied with our Golden Gate Assembly kits.
The tool will help design PCR
primers to make amplicon inserts,
check sequences for internal type IIS
restriction enzyme cut-sites, and
generate a report that shows the assembly
graphically and includes a full
construct sequence file.
Begin by selecting the type IIS
restriction enzyme you have chosen
for your assembly design from the drop
down menu.
Enzymes available from NEB are shown
in red.
Choose the restriction enzyme that has no
internal sites in the DNA sequences
to be used in the assembly.
If this is not possible, view
our video on Golden Gate domestication
for strategies dealing with internal sites.
In our example, we will select
BsmBI-v2
and use sequences that have no internal
sites.
Add the destination vector.
Add the destination vector by clicking
on add sequence.
Select use sample sequences.
Then, select PGGa select
from the destination vectors
drop down menu.
The sequence will appear in the parse sequence
window, and the vector name will be shown.
If you are loading your own destination
vector sequence, you can paste
it in manually or load
your GenBank, FASTA, or
raw sequence file.
You will also have the option of using the sequence
or converting it to its reverse
complement to fit in the correct orientation
in your desired final assembly.
Note, the destination vector has been identified
as your vector and as circular.
Next, select existing
BsmBI-v2 cut-sites.
Since PGGa select already
has design flanking BsaI
BsmBI,
and BbsI sites at the
assembly site of the plasmid. For
other proposed destination plasmids,
the tool will warn you if sites are not
present or if they are improperly
oriented.
The graphics should now show the PGGa
select vector backbone.
Click on add.
Now we will add the first insert.
Click on add sequence.
Select
use sample sequences.
Select insert one from
the sample inserts drop down menu.
The sequence will now appear in the parse
sequence window and the insert name
will be shown.
Notice, the vector and circular identifiers
have not been checked, as the
entire sequence in the sequence window
should be the desired insert.
Next, select PCR
generated
BsmBI-v2 sites
To automatically build flanking
BsmBI sites in the primer designed
for your insert PCR.
If loading your own insert sequence,
you have the option of designating the beginning
and end of the desired insert sequence
by entering the start and end base
numbers from your uploaded sequence.
You can also generate the reverse compliment
if this is required for the correct orientation
of your building assembly.
If you're insert sequence is part of
an uploaded plasmid construct, it
is important to identify the uploaded sequences
being circular,
in case you're insert spans the beginning
of the presumed linear sequence file.
For our demonstration, however, we
will use the entire sample insert
sequences
in the appropriate orientation as shown.
Click on add.
As you can see,
the graphic now shows both the PGGa
select vector backbone
and the first insert.
This button will be disabled if the program
detects an internal site in your insert
sequence.
Add the 2nd 3rd 4th
and 5th inserts in a similar way.
Repeat the steps above for each
fragment you would like to add.
In this demonstration, we will add
inserts 2, 3, 4,
and 5. Always remember
to click on add for each added
insert.
With each addition, the graphic will show
the added insert on the map
with the order of inserts shown above.
After all inserts are added, the
graphic shows the PGGa select
vector backbone in all inserts.
You have now indicated your chosen
destination plasmid vector backbone
and the five inserts in the desired
order. At this point,
you can review your planned assembly.
Are the inserts in the desired order?
If not,
rearrange an insert by selecting it
and dragging it to its new position.
Check the notes section for any warnings
that should be assessed before moving forward.
These warnings differ in severity.
Note, the strongest warnings indicate issues
that could lead to assembly failure.
These include:
self annealing primers that could generate
a Golden Gate active false insert
during PCR,
an overhang that our T4 DNA
ligase fidelity research identifies
as leading to poor assembly efficiency,
or
a discontinuity or lack
of a proper overhang between assembly
sequences.
For more details on warnings, please
refer to the help section of the tool.
Is the estimated legation fidelity
high enough to ensure assembly success?
If not,
click on optimize or
refresh to receive additional recommendations.
Note, that further information about junctions
is available by selecting any insert
in the graphic.
This will bring up a color coded listing
of the insert with both its upstream
and downstream regions.
Also displayed will be the suggested
primers for each amplicon insert
to be generated by PCR
with 5' flanking bases
and the chosen type IIS restriction
enzyme recognition site
added to each primer.
Also listed, are the recommended
oligonucleotide primers with their length,
%GC, and suggested
annealing temperatures.
Next,
click on the project summary tab
for a full assembly report that will
contain all your information.
You can export a PDF of the assembly
by clicking on export summary.
PDF.
To export component fragments,
click on export fragments
FASTA.
You have the option of listing your suggested
primers
in an IDT compatible format
or a FASTA format.
Click on export oligos
to generate a text file of the primers.
For your assembled sequence, choose
the format you prefer by checking
the box for a FASTA,
GenBank, or serial
cloner format,
and click on export assembled
sequence to export.
Review the assembled color coded sequence
for your completed planned assembly.
Click Done.
Other useful options within the assembly
tool include
the Preferences Tab, which indicates
the ligation fidelity data set
used to generate high fidelity overhangs
for your chosen typeIIS restriction
enzyme
to direct the assembly.
The PCR DNA polymerase
or kit,
used to estimate the annealing temperatures
for the generated PCR primers.
NEB recommends the use of the
high Fidelity Q5 DNA
polymerase
to minimize errors generated during
PCR for amplicon inserts.
But, other options are available in
the drop down menu.
You can change any of these parameters,
just be sure to click save changes
when done.
The load/ save project tab
allows you to rename your assembly
project, if desired.
You also have the option to save
it to your computer or load it
at a later time for further analysis.
The Golden Gate utilities tab
allows access to the three
associated, browser based ligase
fidelity web tools.
The view overhang set fidelity
shows the predicted ligation fidelity
for your generated overhangs.
The get high fidelity overhang
set uses T4 DNA
ligation fidelity data generated
from research performed in New England Biolabs
to provide lists of overhangs
known to ligate by this enzyme with
high fidelity.
The split sequence for scarless assembly
will examine an uploaded desired
full assembly and divide it into
inserts based on your stated number
of fragments.
Please note that for assemblies with greater
than 20 fragments, we suggest you
use the ligase fidelity tools
on our NEBeta tools webpage.
The question mark icons indicate
further information is available
about various steps while using the
web tools.
We hope you find the assembly web tool
useful,
and, as always, we are available
at info@neb.com for
any questions