A couple of questions

How do you set up a restriction digest?

Set up the reaction using the following scheme: 1) Determine the amount (total ug and total ul) of DNA to be digested. 2) Use the ug amount of DNA to determine how many enzyme units to use. 3) Determine how many ul of enzyme to use, using the enzyme concentration. 4) Choose a total volume for the reaction.

How is restriction digest done?

Restriction digestion is accomplished by incubation of the target DNA molecule with restriction enzymes — enzymes that recognize and bind specific DNA sequences and cleave at specific nucleotides either within the recognition sequence or outside of the recognition sequence.

How do you store a restriction digest?

The product of restriction digestion can be easily stored at -20 C. At 4 C it would be fine but to ensure that there is no activity and no star activity it is recommended to keep it at -20 C.

How do you add restriction sites to plasmids?

A restriction fragment can be inserted at a restriction site in the vector, or it can replace a restriction fragment in the vector.

  1. Specify the Insertion Site. …
  2. Open the Insert Fragment Dialog. …
  3. Preview the Vector. …
  4. Specify the Fragment Source Sequence. …
  5. Specify the Insert Fragment. …
  6. Preview and Create the Product.

How much BSA do you add to restriction digest?

3 µL 10x BSA (if recommended)

How do you know if your restriction digestion was successful?

If the digested product would be visible at a lower coordinate on the gel, it would have made things easy. You can amplify your digested fragment with primer beginning in the flankers region and with only 3-4 bp in the intern 8680 bp region. If you do not get PCR fradments, was the digestion successfully.

How much DNA do you put in restriction digest?

In general, we recommend 5–10 units of enzyme per µg DNA, and 10–20 units for genomic DNA in a 1 hour digest.

Can I store restriction enzymes at?

Storage at -20°C is recommended for most restriction enzymes. For a few enzymes, storage at -70°C is recommended for periods longer than 30 days.