A 100 – 200 ng plasmid DNA is sufficient to analyze by restriction digestion. Here we describe a procedure to double digest plasmid DNA with FastDigest EcoRV and HindIII restriction enzymes (from ThermoFisher Scientific). This procedure can be used to check the presence of a DNA fragment cloned in a vector. Double digestion will release the fragment of the appropriate size.
Equipment and disposables
Microcentrifuge tube (0.5 ml or 1.5 ml) (DNase-free, autoclaved)
37°C water bath
Reagents and solutions
FastDigest EcoRV (Eco32I) (Therofisher, #FD0303)
FastDigest HindIII (Therofisher, #FD0504)
10X FastDigest Buffer/FastDigest Green Buffer
DNase-free water (e.g., Therofisher, #4387936)
Double digestion of plasmid DNA with FastDigest EcoRV and HindIII restriction enzymes in a total reaction volume of 20 µl
Prior to start:
Set the water bath at 37°C
Step 1: Set the reaction as follows
|Plasmid DNA (100 ng/µl)||2 µl|
|FastDigest EcoRV||1 µl|
|FastDigest HindIII||1 µl|
|10X FastDigest Buffer||2 µl|
|Nuclease-free water||14 µl|
|TOTAL VOLUME||20 µl|
1. Use the following order to add reaction components: Water (first reagent to be added in the tube) → 10X FastDigest Buffer → Plasmid DNA → FastDigest EcoRV/HindII enzyme (the last reagent to be added in the tube)
2. If plasmid DNA concentration is different, adjust the water volume according to DNA volume. For example, if the DNA concentration is 50 ng/µl, you will add 4 µl DNA in order to add 200 ng DNA to the reaction. In this case, you need to take 12 µl water to the reaction to make the final reaction volume 20 µl.
3. The plasmid concentration in a reaction can varies from 100 ng to 500 ng in a reaction. The lowest amount depends on the sensitivity of detection method on the agarose gel.
Step 2: Incubate tube (reaction mix) at 37°C for 15 min.
Step 3: Analyze the digested plasmid DNA by agarose gel electrophoresis.