Setting up the Ligation Reaction
The amounts and concentrations of the components of the ligation reaction are not written in
stone. If possible I try to follow the suggestions of whoever I purchase the enzyme (ligase)
from, because they usually go through some effort to make sure their recommended
conditions work. HOWEVER, lots of times you can not do that (maybe your insert
concentration isn’t high enough, or you don’t have enough vector or whatever), and you need
to fudge some – its OK. This is not a very precise procedure.
From the product information sheet, since we are using cohesive ends, we should aim for
Reaction volume:
Total DNA:
Insert:vector [molar] ratio:
20 uL
10-100 ng
3:1
Also given are a recommended number of fmol of vector and insert. It is not usually
necessary to calculate these (for vectors and inserts of typical size, the fmol will be in the
right range if you hit the ng target), but they are given because units of ligase is defined in
terms of “moles of substrate converted’ and the insert and vector ends are the true substrates
for this enzyme)
Lets plan to use 50 ng of vector (just arbitrary).
How much insert is needed?
NO, its not 150 ng!!! – remember that it is a molar ratio!
You can go through an entire complicated dimensional analysis to figure this out in actual
moles (using 660 as the mw of one base pair), but it is far easier to use proportions.
First, estimate approximately how much you will need (this way when you figure it out in
real numbers, you will know if your answer is right).
Keep in mind that 1 molecule that is 300 bp will weigh less than a molecule that is
3000 bp. In fact, it will weigh about 10X less. So, if your vector is, say 2700 bp,
and your insert is 270 bp, if you use 50 ng of vector, for a 1:1 molar ratio you will
need 5 ng of insert, and thus, for a 3:1, 15 ng.
OK now having done that, lets say that the insert was 209, and the vector was 3645.
(Remember, this is the vector you are going into – the new receiving vector)
First determine the size ratio, 290/3645 =
X
Then multiply 50 ng (the # ng of vector you will use) x X = # ng of the small insert
fragment that will contain the same number of molecules (or moles) as you would
have in 50 ng of vector; i.e., a 1:1 molar ratio.
To make a 3:1 insert:vector ratio, x3 = ______
Now do this calculation for the insert (209 bp) of interest and your vector (pUC 19)
Now you can go back to the quantification on the insert, and figure out how much volume
this is. (note that the precision of measurements here is limited by the precision with which
you can quantitate small volumes of DNA. No point in being overly compulsive, just get
close!)
Now in this case, we have one more thing to consider. We calculated for the 209 bp
fragments, but in most cases our preparation actually contains two fragments, both of which
are ligatable in our system (b/c both have one EcoRI end and one HindIII end). If we put in
the 209 bp at a 3:1 ratio, we will actually have a 6:1 ratio of ligatable fragments (b/c we will
have the same number of 250s present also). Too many inserts per vector can increase the
chances that you will get concatamers (in this case, three inserts --vector-H H-E E-H HE E-vector ----). Since the 209 and 250 are themselves in a 1:1 molar ratio, it is appropriate
to use just half of whatever ng amount (or volume) that you had calculated for the 209s
alone, because you will be adding also an equal number of 250s. (Remember, the ligase does
not care whether its 209 or 250!)
Setting up the reaction:

Once you have worked out the volumes needed for insert and vector, you only need to
put it all together.

Try to hit the 20 ul volume.(remember that the buffer for ligase is 5X rather than 10X)

Note that the enzyme amount for cohesive end cloning is 0.1 U, but the concentration is 1
U/uL (you will indeed have to dilute the enzyme -- water is OK, as long as you don’t do
it more than a few minutes in advance – 1/10 (record the actual volumes for the dilution
in your lab book). Do keep the volume of your dilution small (use no more than 2 uL of
the concentrated enzyme) and share with a partner, so as not to waste it). This does make
a difference. I don’t know why.
HOWEVER – if you have compatible ends, you can forgo the dilution, use the enzyme at 1
unit/rxn and do it in 5 minutes. (Invitrogen says that you should also use “MaxEfficiency”
Competnet cells, which we do not have. But I suspect it will work anyway.)
We might want to try some different conditions, and we definitely want some controls (see
below), so we need to plan these, but first need to determine ….
How many ligations can each student do? (for this we need to look ahead)
In normal practice, you can do as many as you want, but here we are somewhat limited by
the supplies – ultimately each ligation needs to be transformed (into competent bacteria), and
plated, so we need to figure out how many transformations we can do.
The competent cells come in vials of 0.5 mL. Once they are thawed we do not freeze them
back – they are too fragile. So we will try to make optimal use of a few number of tubes, and
still get the job done.
The standard transformation is done with 50 uL, but some people use less (25 uL). So we
can get 10 – 20 ligations per tube. Each pair should use cells from the same tube, to
eliminate tube-to-tube variation. Plan to do 5 transformations per pair.
Now in addition to the transformations of ligation reactions, we need to do one or two
transformation controls. We would like to know the competent cells are good (our ligation
reaction could be perfect, but if our competent cells failed, how would we even know?), so
we transform with:
 PUC19 provided by the manufacturer of the competent cells (tells you transformation
efficiency
So…if each pair can do 5 transformations; one of intact pUC 19 and four from ligations.
plus 4 ligations we would do the following:
L1
3:1 insert to vector
L2
0:1 (vector only) + ligase
L3
pick from the options below
L4
pick from the options below
(what are we checking for here?)

0:1 (vector only) no ligase (checking for what?)

3:1 different source of insert (maybe in-agarose insert, if we have some?)

3:1 different prep of vector (that of another student, or QIEX vs phenol vs
heat inactivated?)

ligation control (not using our insert and vector at all, but a plasmid that is
known to be ligatable (singly cut) to test whether the ligase itself worked
And looking ahead just a bit further .. how many plates will each person need?
Figure on 2-3 plates for each transformation. (Do initial with 2, and if you need more for a
second plating, you will have them ready)
So 5 transformations x 3 plates (LB amp) = 15 per pair (x 9 so minimum 135 plates)
(I will request these from the stockroom, but remember to think of this ahead of time, and be
sure you have plates, with the appropriate antibiotic for your vector!)