What does double digest mean?

The product formed when two different restriction endonucleases act on the same sample of DNA.

Why do a double digest?

Digesting a DNA substrate with two restriction endonucleases simultaneously (double digestion) is a common timesaving procedure. Selecting the best NEBuffer to provide reaction conditions that optimize enzyme activity as well as avoid star activity associated with some enzymes is an important consideration.

What enzyme digests the fastest?

It’s that easy. Thermo Scientific FastDigest restriction enzymes are an advanced line of enzymes which offer: Complete digestion in 5-15 minutes. 100% buffer compatibility with downstream applications.

What is FastDigest?

Description. The 10X FastDigest Buffer is a proprietary digestion buffer which supports 100% activity of all FastDigest restriction enzymes. DNA/RNA modifying enzymes, such as ligases, phosphatases, kinases and mesophilic DNA polymerases also have 100% activity in FastDigest Buffer.

Is single or double digest better?

The recombinant fragments of single-digested plasmids have to be selected for their proper orientation while the double-digested plasmids ensure the proper orientation of the foreign DNA fragment. Therefore, double-digested plasmids save time in the recombinant DNA techniques, not single-digested plasmids.

How do you know if you are not digesting protein?

Symptoms of protein malabsorption include indigestion, gas, bloating, acid reflux, GERD, constipation, diarrhea, malabsorption, nutrient deficiencies, hypoglycemia, depression, anxiety, trouble building muscle, ligament laxity.

What is the easiest protein to digest?

Here’s a list of some easy to digest proteins and how to prepare them to get your gut back on track.

• Light, Flakey Fish. Because white fish is low in fat and fiber-free, it is one of the best sources of high-quality protein and easy on your gut.
• White Meat Chicken and Turkey.
• Eggs.
• Milk.
• Tofu.

What is in FastDigest buffer?

The 10X FastDigest Green Buffer includes a density reagent and two tracking dyes for direct loading. The blue dye migrates with 3–5 kb DNA fragments in a 1% agarose gel and has an excitation peak of 424 nm. The yellow dye migrates faster than 10 bp DNA fragments in a 1% agarose gel and has an excitation peak of 615 nm.

What is double digestion with restriction enzymes?

A double digest is one where two restriction enzymes are used to digest DNA in a single reaction. In this case you will be using EcoR I and BamH I. There is only one site in the plasmid vector for each of these enzymes and they are located on either side of your insert DNA.

How much plasmid do you need for digestion?

*Pro-Tip* Depending on the application and the amount of DNA in the reaction, incubation time can range from 45 mins to overnight. For diagnostic digests, 1-2 hours is often sufficient. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least 4 hours.

How long can you leave a restriction digest?

Time-Saver qualified enzymes can cut substrate DNA in 5-15 minutes and safely digest overnight. For enzymes that are not Time-Saver Qualified, the recommended incubation time is 1 hr. In general, long incubations (several hours to overnight) are not recommended, unless digesting some gDNAs.

When to use a pre stained protein ladder?

Prestained ladders may be used for molecular weight approximation, confirming gel migration and estimating blotting efficiency but for accurate molecular weight estimation, an unstained ladder should be used. Can I use any of your protein standards for estimation of protein quantity (protein quantitation)?

How are protein markers used in the western blot?

The protein markers consists of recombinant proteins with an IgG binding site. The IgG binding site binds the primary or secondary antibody used for detection of the target protein, allowing visualization of the standard on the western blot.

When to use FastDigest buffer with no star activity?

Short protocol times with no star activity FastDigest enzymes in combination with FastDigest (Green) Buffer are designed to eliminate star activity: Short incubation time (5–15 min), no need for prolonged digestions. Universal buffer with optimal pH and salt concentration for all enzymes.

How are FastDigest enzymes used in Golden Gate cloning?

By addressing all these issues, FastDigest enzymes enable single, double and even triple digestion in 5 minutes without any signs of star activity. FastDigest EcoRI and BamHI restriction enzymes were used to prepare vector for Golden Gate cloning method.