Reading gels and colony plates

A result is only as good as its control

A gel with bands and a plate with colonies are not an answer. They are data. To read them, you need a ladder and some controls. This lesson shows how.

Reading a gel with a ladder

In the first course you used a model in which the distance run is a straight line against the log of the size. A real gel works in the same way. The ladder is a lane of fragments of known sizes. You measure the distance of each ladder band from the well. Then you plot the log of the size against the distance. The points lie near a straight line. You draw the line, or you fit it with a short program. Then you read the size of any unknown band from its distance.

The reading is good inside the range of the ladder. Outside it, the line is not tested, and the reading is a guess.

The forms of a plasmid

An uncut plasmid can show more than one band, though it is one DNA. The reason is its shape.

• The supercoiled form is tightly twisted and compact. It runs the fastest.
• The linear form has been cut once. It runs at its true size.
• The nicked, or open circular, form has one strand cut. It is loose and runs the slowest.

So a plasmid cut once with an enzyme shows one band at its true size. This is a quick check that a digest worked. A plasmid cut at $k$ sites gives $k$ bands, if the pieces differ in size. Two equal pieces run together as one thicker band. Add up the sizes of the bands. The sum must be the size of the plasmid. If it is not, a band is hidden or a size is wrong.

Reading a colony plate: the controls

A transformation can fail in many ways. Control plates tell you which. Here is a standard set.

If the plate with no DNA has colonies, the antibiotic is not working, or the cells are contaminated. If the uncut vector gives few colonies, the cells are not competent, and every other plate is useless. If the cut vector without ligase gives many colonies, the digest was incomplete.

Transformation efficiency

The efficiency of a batch of competent cells is the number of colonies for each microgram of plasmid DNA. Only the part of the cells that you plate counts. If you used 2 ng of DNA and plated one tenth of the cells, only 0.2 ng of DNA was on the plate. With 184 colonies, the efficiency is $\dfrac{184}{0.2\ \text{ng}} = 920$ colonies for each nanogram. That is 920000 colonies for each microgram. Cells that are made in a teaching laboratory with calcium often give $10^5$ to $10^7$, and commercial cells give $10^8$ or more. These are typical values, and NCERT does not give them.

Blue and white, a little deeper

The lacZ test of the last course uses two chemicals. X-gal is the chromogenic substrate: the enzyme breaks it into a blue product. IPTG switches on the promoter of the lac genes, so that the enzyme is made. A white colony is not always a recombinant: a small share carries a vector with a damaged lacZ, or no insert. So the white colonies are checked by a digest or by PCR.

You should see that the efficiency is proportional to the number of colonies. Plating a smaller part of the cells does not change it, because the DNA on the plate falls in the same proportion.

Try it in Python

The program calculates the efficiency, and the shares that the control plates show.

Try the ladder in Python

The next program fits the ladder and reads three unknown bands.

Practise with new numbers

Common mistakes

• Reading a size outside the ladder. The line is not tested there.
• Calling the faster band of an uncut plasmid a smaller plasmid. It is the supercoiled form of the same plasmid.
• Counting all the DNA in the efficiency. Only the DNA on the plate counts.
• Ignoring the control without ligase. It tells you whether the digest was complete.
• Taking every white colony as a recombinant. Some are not.

Remember these

• A ladder with a straight-line fit of log size against distance gives the size of a band.
• Uncut plasmid: supercoiled runs fastest, linear next, nicked slowest. The sizes of the pieces of a digest add up to the size of the plasmid.
• Controls: no DNA, uncut vector, cut vector without ligase, cut vector with ligase and no insert.
• Efficiency is the colonies for each microgram of DNA that was on the plate.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Why is a ladder run next to the sample?

2. A plasmid that is cut once with an enzyme gives how many bands, and where does it run compared with the uncut form?

3. What does it mean if the plate with no DNA shows colonies?

4. The plate of the cut vector without ligase has colonies. What does it show?

5. 1 ng of DNA is used, and 50 per cent of the cells are plated. 250 colonies grow. What is the efficiency in colonies for each microgram?

References

• Sambrook J. and Russell D. W. (2001). Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, chapters 1 and 5.
• Brown T. A. (2016). Gene Cloning and DNA Analysis: An Introduction, 7th edition, Wiley, chapters 5 and 6.