I have performed restriction digestion with ScaI for plasmid DNA. By insilico analysis, I saw that there is only one site for ScaI in my plasmid. So in normal conditions, I should get only one band on the agarose gel, but, I am getting 2 bands. WHY?
possibly cut and uncut plasmid running at the expected different sizes. A picture would help to get an accurate answer and also the amount of enzyme,template dna and time of the cutting reaction to determine the likelihood of a partial digest
Its important that you include the undigested plasmid in the same gel as reference.
5' A G T ↓A C T 3' 3' T C A ↑T G A 5' Thermo Scientific ScaI restriction enzyme recognizes AGT^ACT sites and cuts best at 37°C in its own unique buffer. See Reaction Conditions for Restriction Enzymes for a table of enzyme activity, conditions for double digestion, and heat inactivation for this and other restriction enzymes. Note: Also available as a FastDigest enzyme for rapid DNA digestion. Isoschizomers: AssI, BmcAI, ZrmI.
Thermo Scientific conventional restriction endonucleases are a large collection of high quality restriction enzymes, optimized to work in one of the buffers of the Five Buffer System. In addition, the universal Tango buffer is provided for convenience in double digestions. All of the enzymes exhibit 100% activity in the recommended buffer and reaction conditions. To ensure consistent performance, Thermo Scientific restriction enzyme reaction buffers contain premixed BSA, which enhances the stability of many enzymes and binds contaminants that may be present in DNA preparations.
in the gel image... 2, 7 and 12 lanes are uncut plasmid whereas...4, 9 and 14 lanes are plasmid cut using ScaI.
Enzyme added was 0.5 micro litre and incubation time was 3 hours at 37°C.
Paul Rutland Hey Paul, For reference I have included the gel image. Kindly check.
Thank YOu
Hi Simant Kumar .
It looks like a partial digest to me. Try incubating overnight with .5 ul enzyme and also using 1ul of enzyme. Use a control dna (genomic or lambda dna in a separate reaction to check that the enzyme is capable of cutting and has not become inactive
It is possible that the buffer being used for the reaction is not at an optimum concentration. You can try to look at the dilution of the buffer while setting up the reaction. Also, it is possible that there is not enough enzyme for the amount of DNA being added. To calculate the optimum amount of enzyme you can try looking at the information of how many enzyme units can digest how much amount of DNA. This is available on the enzyme's information page.
Apparently incomplete hydrolysis of plasmid DNA. It is necessary to increase the incubation time and possibly the amount of added enzyme.
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