NEBCalculator is an easy
to use tool that contains various
modules to help with biomass calculations.
In this video, we will demonstrate
how to use the double-stranded DNA
mass to from moles
converter to plan and NEBuilder HiFi
DNA assembly reaction.
The double-stranded DNA mass
to from moles converter module
has two modes. The mass
to moles mode converts the double-
stranded DNA mass to
moles of double-stranded DNA,
moles of double stranded DNA
ends,
and the number of DNA molecules.
The moles to mass mode converts
moles of double stranded DNA to
the DNA mass.
This can be useful for setting up in NEBuilder
HiFi DNA assembly reaction.
NEBuilder HiFi DNA assembly
efficiently assembles 2 to
6 fragments of double-stranded
DNA in a single reaction.
An important parameter of any DNA
assembly reaction is the number of
DNA molecules present in the assembly.
NEBioCalculator can help convert
DNA mass concentration to moles.
For a 2 to 3 fragment assembly,
NEB recommends using a total
DNA quantity of 0.03
to 0.2 picomoles
and a 1 to 2 vector to insert
molar ratio.
We recommend starting with 50 to 100
nanograms of vector fragment
when planning a reaction.
You may need to use more DNA mass
when working with large vectors. In
this example, we demonstrate a
three fragment assembly, one
vector fragment and two insert fragments.
The linearized vector is 3 kb
in length and the insert fragments are
500 base pair and 2 kb,
including the assembly overlaps.
We will first determine how much
of the vector should be added to the assembly
reaction.
Start with the recommended 100
nanograms of vector to begin our
calculations. Click on the double-
stranded DNA mass
to from moles conversion button.
Click on the master moles tab.
Enter 3 kb for DNA
length and 100 nanograms
for the DNA mass. Make sure
you select the correct unit type from
the pull down.
NEBioCalculator tool immediately
calculates that the number of moles
of vector is 53.94
femtomoles or 0.0539
picomoles of vector. We can
then use this to determine how much
insert DNA to use. For
the inserts, we need a 1 to 2
vector to insert ratio. Multiply
0.0539 picomoles
of the vector by two
to get
0.108 picomoles
of each insert fragment.
It is important to stay within the total
DNA recommendation for the assembly
reaction or 0.03
to 0.2 picomoles total.
If we sum the total DNA for our fragments,
you can see that we have greater than
0.25 picomoles of
DNA, which is too much.
If we repeat our calculations with
less vector DNA, for example
75 nanograms, we
find that we have 0.04
picomoles of vector, and
0.8 picomoles of our two
fragments for total DNA input
of 0.2 picomoles,
which is within the recommendations.
You may need to use a larger starting mass
when working with large vector fragments
greater than 6 kb.
Now that we have the correct quantities
of DNA fragments to add to our assembly,
we can use the insert fragment lengths
to calculate the mass of each insert
DNA to add to the NEBuilder assembly
reaction. By entering fragment length
and moles, NEBioCalculator
indicates we should add 24.72
nanograms of the 500 base
pair fragment
and 98.87
nanograms of the large 2 kb fragment.
With this information, you can start setting
up your NEBuilder HiFi DNA
assembly reaction.
Visit NEBCalculator.neb.com
for more
helpful bio math modules and
NEBuilderHiFi.com.
for more information on NEBuilder
Hi Fi DNA assembly.