BioBrick Assembly Standards and Techniques and Associated Software Tools - DNA Cloning and Assembly Methods - page 18

After adding Buffer EB, increasing the incubation time up to
4 min can increase the DNA concentration.
10. Measure the concentration using a NanoDrop [ 17 ] and write
the concentration on the tube. Store at −20 °C.

The presented protocol is from partsregistry.org [ 18 ].

3.7 Ligation

1. Use Eq. 1 to find volume of backbone and volume of insert to
be used. Total volume (backbone + insert) should be 15 μL,
and normally, the ratio 1:3 between backbone and insert can
be used.

size CVn
size

× × ×

insert backbone backbone

V = (1)

insert

× C

backbone inse rt

2. Make two ligation samples in 1.5 mL tubes; one with the insert
and one with dH 2 O added instead of insert (the latter is called
a religation and is used as a control experiment).
3. Add 2 μL of T4 DNA Ligase Reaction Buffer.
4. Add 1 μL of T4 DNA Ligase, mix, and spin down.
5. Incubate for 30 min at 16 °C and 20 min at 80 °C to heat kill
the enzymes.
6. Store at −20 °C, or use 2 μL of the ligation mixture for trans-
form to competent cells.

In this expression, the sizes of the insert and backbone are
measured in base pairs, concentration is measured in ng/μL, and
volume is measured in μL. n denotes the ratio between insert and
backbone, with respect to the insert. For example; if three times as
much insert as backbone is used, then n = 3.

3.8 Making a
Construct of Two Parts

1. Obtain plasmids with the genes of interest, many can be
found through the Registry of Standard Biological Parts
( http://partsregistry.org/Catalog ) .
2. Decide which should be treated as insert and backbone
( see Note 7 ), and perform a restriction digestion as described in
Subheading 3.4 . Enzymes and buffers to be used can be seen
in Table 1 .
3. To obtain the correct pieces of DNA, investigate the fragments
resulting from the restriction digestion using gel electrophoresis
(described in Subheading 3.5 ), followed by excision of the cor-
rect gel piece, and gel purification (described in Subheading 3.6 ).
4. Ligate the two pieces together, using the protocol for Ligation
(described in Subheading 3.7 ) ( see Note 8 ).
5. Use 2 μL of the ligation mix to perform a transformation
(described in Subheading 3.1 ).