TITLE: Keller's Band Counting Method with Chloroquine
AUTHORS: Salima Rüdiger, Rainer  Machne

[DESCRIPTION]
Supercoiling plays a major role in the regulation of bacterial growth, especially in the circadian rhythm of cyanobacteria. The Keller's Band Counting Method was modified with the use of intercalating substance chloroquine (CQ), instead of using ethidium bromide (EthBr). The principle of that method is to conserve supercoils through intercalation and unwinding of not intercalated supercoils through Topoisomerase I. Using different concentrations of the intercalating substance cause different levels of supercoiling. The treated samples were applied on agarose gels containing different concentrations of intercalation substance as well. This method allows counting all topoisomers from native, to completely relaxed state. So, the full extent of supercoiling is defined exactly. The modified method can be adapted easily, to establish correct conditions to monitor the in vivo supercoiling of other, biotechnologically relevant plasmids (expression vectors of reporter genes or heterologous expression).

[STEPS]
SECTION: Isolation of plasmid
1. The examined plasmid has to be isolated properly.
For a bigger topoisomer series, it is recommended to isolate a higher amount of bacterial culture. 
I use ZymoPURE™ Plasmid Maxiprep Kit, according to their Vacuum protocol. 
SECTION: Purification with T5 Exonuclease (optional)
2. The isolated plasmid sample is divided into 1 µg aliquots to perform the T5 Exonuclease reaction. The concentration was measured with Nanodrop. 
 
Each reaction contained: 
1 µg DNA
5 µl NEBuffer 4
1 µl T5 Exonuclease
fill up to 50 µl with Nuclease-free water
Incubate at 37 °C for 30 minutes.
 
This protocol is based on the protocol of NEB.  
SECTION: PCR clean-up/purification (optional)
3. To purify plasmid samples from the T5 Exonuclease a PCR purification should be deployed. Summing up samples in one column helps to get higher concentration in single samples. It is important to not press the threshold of 10 µg per column to not lose any DNA. 
For this step, I use the NucleoSpin® Gel and PCR Clean-up kit from Machery-Nagel according to their PCR clean-up protocol.
SECTION: Plasmid treatment for "Keller’s Band Counting Method"
4. At first, the plasmids get in contact with different concentrations of chloroquine:
1 µg pDNA
2.5 µl Cutsmart 
chloroquine 0-3 mg/ml (calibrated for pUC19)*
fill up to 24 µl with Nuclease-free water
Incubate at 37 °C for 15 min
 
After that, add 1 µl of Topoisomerase I in each reaction. 
 
Incubate it again at 37 °C for 15 min. 
Then stop the reaction at 65 °C for 20 min. 


*It is easier to prepare a chloroquine dilution series, where you just put e.g. 5 µl of the mixture to get your desired concentration in your reaction


 
SECTION: Chloroquine agarose gel electrophoresis (pUC19)
6. This section is calibrated for pUC19 and the
Peqlab chamber 40-1515: 
 
At first, a 1.2% agarose gel is needed: 
225 ml 0.5x TBE buffer
2.7 g agarose 
Boil the mixture in the microwave and when it is warm to the touch add chloroquine (end concentration 0 - 8 µg/ml) in it.

Then the fluid gel is ready to be poured in the prepared gel trail. I use the Peqlab 40-1515 chamber. 
Prepare 1 L of 0.5 TBE buffer with the same Chloroquine concentration as the gel.

When the gel is solid, put the trail into the chamber and 1 pour the prepared TBE buffer in it. 





A detailed chloroquine agarose gel electrophoresis protocol is linked down below: 
 


SECTION: PCR clean-up/purification
5. To purify plasmid samples from the chloroquine and Topoisomerase I, a PCR purification should be deployed. That also helps to reconcentrate the samples with summing similar treated samples into one column (see 3).
For this, I use again the NucleoSpin® Gel and PCR Clean-up kit from Machery-Nagel according to their PCR clean-up protocol.