NEBuilder 2.0 generating
vector by restriction enzyme digestion.
In this video, we will demonstrate
how to use the NEBuilder assembly tool
to build a construct using a restriction
enzyme digested vector
and two PCR generated inserts.
The tool will help to design PCR
primers containing the required overlap
sequences.
We will also regenerate one of the restriction
enzyme recognition sites.
Begin by clicking on the settings
tab.
Choose The assembly product and
the PCR polymerase.
The assembly can be linear or circular.
We will be making a circular assembly
product.
Click done.
Now. We will add the fragments to
the assembly.
Begin by clicking on build,
then click
new fragment.
For this demonstration,
we will use preloaded sample sequences.
Note that all vectors available from NEB are
pre loaded into the tool.
Click Use sample sequences.
Select a cloning vector from the drop
down menu. In this case, pUC19.
The selective sequence is now loaded in
the sequence field.
Make sure that you check both vector
and circular.
In step two, name your fragment.
In step three, select a method
of production.
In this example, we're using a
fragment produced by restriction enzyme
digestion.
Now select the restriction enzymes that you
will use to digest the plasma from the drop
down menu.
We have chosen HindIII and EcoR1.
Note that only restriction enzymes with one
or two sites in the plasmids are available.
By convention, the insert fragment
will be cloned in the clockwise direction
with the 5' end of the insert
joined to the 5' end of the vector,
and the 3' end of the insert
joined to the 3' end of the vector.
In this case,
The upstream 5' site
is EcoR1 and the downstream
3' site is HindIII.
Once you have selected the restriction enzyme
sites, the map in the upper right
will show the digested plasmid.
You can use the swap button to
change the upstream and downstream
sites.
Notice how the plasmid map changes
when the restriction enzyme sites are swapped.
Click add.
On the next page, you can edit the fragment.
Click the pencil icon to edit the fragments
properties or add a spacer.
We will edit the fragment by regenerating
the HindiIII site.
Site regeneration adds bases to
primers in order to rebuild the Restriction
Enzyme Recognition site.
We're not using primers to generate this
fragment, but the information will be
used in the design of the primers for
adjacent fragments.
Go to the downstream 3' site
and under site regeneration, choose
strict.
Strict regeneration adds all
the bases required to regenerate the restriction
enzyme site with no regard for
the sequence of the adjacent fragment in
the assembly.
Minimum regeneration takes into account
the sequence of the adjacent fragment
in the assembly and will use bases
from that fragment if appropriate,
to regenerate the restriction site.
Click Update to make the changes
in the fragment.
Next we will add insert fragments.
Click
plus new fragment.
We will use a sample sequence.
Select LacZ full from
the drop down menu.
Name the Fragment.
Select PCR as production method.
Be sure that auto generate primers
is selected.
If you prefer, you can use custom primers
instead of having the tool designed primers for
you. Enter the start and
end bases.
In this case, we will use the entire
sequence.
Click add.
On the next screen we will see a simple
map of the plasmid vector and the LacZ
insert.
The 5' end of the LacZ insert
is joined to the EcoR1 site.
The 3' end of the LacZ insert
is joined to the HindiIII site.
Scroll down to see the required oligo
nucleotides.
The sequence and black, indicates the additional
sequence added to regenerate the
HindiIIIsite.
Now let's add another fragment.
Click,
plus new fragment.
We will use another sample sequence.
Select GFP from the drop
down menu.
Name the fragment.
Select PCR as the production method.
Be sure that auto generate primers
is selected.
Enter the start and end bases.
In this case, we will use the entire sequence.
Click add.
On the next screen, we see a simple map
of the plasmid vector and the LacZ
and GFP inserts.
The 5' end of the LacZ insert
is joined to the EcoR1
the 3' end of the LacZ insert
is joined to the 5' end
of GFP and the 3' end
of GFP is joined to the HindiIII site.
Fagments can be rearranged by dragging
and dropping the colored bars on the left
into the preferred order.
You can also delete fragments or edit them
by using the functionalities icons
shown in the colored bars.
Click the bars themselves to view the sequence
of the simply junctions,
primer sequences, and the amino acid
sequence for all six reading frames.
The Notes section provides information
about the characteristics of the primers.
The build settings shows the default
settings used to generate the primers. Use
this to change the assembly product,
minimum overlap size, or
polymerase used for PCR.
Scroll down to the required oligo feild.
Each fragment generated by PCR
has a pair of oligo.
the 5' end of each oligo, seen
in lower case,
contains the overlap sequence for the adjacent
fragment in the assembly.
The 3' end of each oligo,
seen in upper case, contains the PCR
primer portion of the oligo.
The GFP reverse primer also
contains the sequence AGC
TT
which are the bases added to the primer
to restore the HindiIII site. The
TM for each primer and the predicted
annealing temperature are all shown.
the annealing temperature is calculated for the
specific polymerase chosen in the first
page. Click done.
On the next page, there will be a summary of
your assembly.
This summary page contains information
on the fragments, the oligos required
for PCR, which can be shown
in FASTA or IDT compatible sequence
formats,
as well as the TM and annealing temperature
of those oligos.
There is also a simple map of the assembled
product, a sequence in multiple
formats that can be exported, and a
color coded sequence that you can easily
identify the assembly junctions.
Use the load save tab to save
your assembly. Enter the name of the
assembly in the Save Project Field
and export the file to your computer.
To start a new assembly, click build
and then start over.
Click okay to delete the
existing project, and then you can start
your next assembly.