DNA Standards
For estimation of fragment size and quantification
There are basically two kinds of DNA standards. “Conventional” standards are some common
plasmid or phage that has been digested by some enzyme into an array of fragments of useful
size. The other is a collection of fragments of some unit size that are attacched together such
that the many fragments create a series of evenly spaced bands, called a ladder.
Ladders are nice for estimating size, because often they are made in nice clean increments. A
1kb ladder will have “rungs” or pieces that are 1 kb, 2 kb, 3 kb etc, and so will look kind of like a
ruler (only a log-scaled one!). Usually one of the bands is made to be brighter than the others, so
that you can figure out which rung is which. (For digested plasmids or phage, pattern
recognition tells you which is which), Such ladders tend to be kind of expensive, though, and are
less useful for quantitation.
By far the most commonly used DNA standard is “H3” which is the bacteriophage digested
with the enzyme HindIII.

is about 48 kb (48,000 bp) in length. When it is digested completely with Hind III:
the fragments, when separated on an agarose gel, give the familiar pattern shown below.
kb:
23
9.4
6.6
4.4
2.3
2.0
the bands of other samples are compared with those of the
standards to determine the approximate size
(these two bands would be about 2.6 kb and 1.7 kb.)
.56
.125
(usually not visible)
The degree to which the bands are spread out or scrunched together depends on the agarose
concentration (lower % is better at resolving larger bands, higher % for smaller bands. The
reason the small one is usually not visiblel is that H3l is typically run on a 1% gel, and the small
band diffuses).
Although these H3 fragments do not make a nice “ruler” like the ladders do, you can usually
tell approximately the size of your unknown band relative to these standards. If you have to
estimate it more exactly, you can plot the migration of each standard band vs. the size (or better,
the log of the size) and make a standard curve)
We can also use the fragments to quatitiate DNA.
Whatever amount of H3 we load will be distributed among the different fragments in proportion
to their size. There is one molecule of each fragment for every molecule of “total” .
If we load 1 ug of H3, which is about 48 kb long, then …
in the 23 kb fragment we will have 23/48 x 1000 ng, or about 500 ng (approx.),
in the 2.3 kb fragment, we will have about 2.3/48 x 1000 ng or about 50 ng, and so on.
If we load 250 ng, these will be 125 and 12.5 ng respectively.
I generally make standard to be 500 ng/12 uL:
Precut standard often comes 0.5 ug/uL, so a 1/10 dilution is 50 ng/uL (which is 500 ng/10 uL) – when you add the 6X
to prepare it to be a gel sample, you add 1/5 the volume, so 2 uL for every 10 that you have, and your final standard
prep is the desired 500 ng/12 uL . You can make any volume you want with these proportions, and keep a stock to
use as needed
The larger bands are brighter than smaller bands. This is because EthBr intercalates between the
bases, and the longer fragments have more bases for taking up the EthBr. In the smaller bands
there are the same number of pieces of DNA, but each piece takes up less EthBr. The more
DNA (# ng) in a particular band, the more EthBr can be taken up, and the brighter the band.
In a quantitation gel, you load several amounts of the standard, and calculate the amount of DNA
that will be in each of the bands.
Make a table that shows the amount of DNA in each band depending on how much
of the total DNA you load. (This table should be in your notebook at least once).
Total loaded:
if 500 ng/12 uL:
500 ng
12 uL
250 ng
6 uL
125 ng
3 uL
%
23
_____
______
______
______
9.4
_____
______
______
______
6.6
_____
______
______
______
4.4
_____
______
______
______
2.3
_____
______
______
______
2.0
_____
______
______
______
.56
_____
______
______
______
Once you have done this a few time, you will see that in a standard size well
100 ng is a very fat band, somewhat distorted; 5 ng or less can be hard to see
10 – 40 ng are very nice bands – strong and clear.
So when you do a quantitating gel, you would like to aim for 10 – 40 ng, if possible.
The standard X174 Hae III is useful for smaller pieces (and higher % gels). It is the
bacteriophage X174 that has been cut with the RE Hae III. Its fragments are shown below:
Make a table for this standard as you did for the H3Add the fragments to get the total size of
the phage, figure out the proportion (or %) for each fragment relative to the total, and then the
number of ng of each if we were to load 500 ng, 250 ng or 125 ng.
1353
1078
872
603
310
271,281
234
194
118
72
note: these two run as a single band, so add their amounts
One final note about the standards: if you are using a comb with very small (narrow) teeth, you
will need to cut down on the amount you use, because that DNA is crammed into a narrower
space. This is a good thing – it allows you to consume less of your sample (and less standard).
****** So that’s how to make the standard curve ... how about the sample? *******
Quantitating the amount of DNA in a sample is done by comparing the brightness of
staining of a known volume of the unknown to that of some standard band
In the same gel (and it has to be the same gel, because it depends on all the bands being stained
and destained the same way) you run a more-or-less arbitrary amount (uL) of the sample you
wish to quantitate.
It is very helpful to estimate much DNA you think will be there, and then how much (volume)
you need to load to see it.
Example: (try this problem. The answer is on the back of this page. Make sure you know how
to figure this out before continuing)
We digest 5 ug of pCAT-P. The plasmid is 4506 bp, and out piece is 209 bp. We clean up our
insert on QIAEX, and end up with 30 uL. Assuming 50% loss, what is the concentration of
DNA in the insert prep?
You should have: 232 ng/30 uL – about 8 ng/uL with no loss, so about 4 ng/uL if 50% loss.
This is what you think you should have, but what do you really have? You need to “spot it on a
gel” (lab slang) and compare it with bands of known DNA amount.
How will you make the sample? You would like to try to load between 5 and 20 ng. So here
that would be about 1-4 uL It might be good to add even more, just in case. I try to load two
different volumes, 5X apart in volume, with the lower volume assuming little loss, and the larger
volume assuming more loss. In this case, I might aim to load 1 and 5 uL (that way if my loss is
modest, I will 4-8 uL at the low end, and should be able to see that.. If I recover only 10%
instead of 50%, my final concentration will be about 0.8 ng/ul, so loading 5 uL should give me 4
ng, and I should be able to see that.
(note: its important to not consume all of your sample doing the quantitation!!
How can you do this easily?
Take 15 uL of the sample. Add 3 uL uL 6X (total 18 uL). Mix and “pop” down.
Load 2.4 uL and 12 uL

that’s equivalent to 2 and 10 uL of the original sample, but with 6X added in).

the reason you make more than 14.4 uL is that you seldom get back all of the volume
you put into a tube, so you need to put in a bit more than you want to get out.)
Analysis:
Compare the brightness of your band to the set of standards – and find a band that more or less
matches it. (It does not have to be at the same size, though that often helps, because the
sharpness of the bands is not the same through the whole gel
When you see the gel, perhaps the band in the 12 uL gel sample (equivalent to 10 uL of the insert
prep) looks the same as a standard band that you know is 5 ng. That means that the
concentration of DNA in your insert prep is 5 ng/10 uL, or 0.5 ng/uL.
Isn’t that easy?
(in loving memory of Jack Herem)