Question: I usually use the supplied EcoRI buffer for EcoRI/SpeI double digests, as recommended in your catalog. However, I always find that this digest is quite inefficient, and I noticed in your current catalog that you report an activity of 25% for SpeI in the EcoRI buffer.

They even invented a new activity unit for these enzymes (Fast Digest Unit: 1 FDU cuts 1 µg control DNA in 5 min in FastDigest buffer). Thermo's FastDigest buffer is probably very similar to NEB's CutSmart buffer For the time being, I just decided to stop buying FastDigest enzymes.

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However, I always find that this digest is quite inefficient, and I noticed in your current catalog that you report an activity of 25% for SpeI in the EcoRI buffer. Choose a DNA, RNA, genome editing, qPCR calculator from NEB, a leader in production and supply of reagents for the life science industry. I did a double digestion of ~2.5µg pET-11a with NdeI and BamHI (both from NEB), followed by a dephosphorylation with Antarctic Phosphatase. Upon running a gel, I noticed that there were two distinct bands in the doulbe-digest (see picture). NEBcutter - On-line DNA restriction mapper tool. This tool will take a DNA sequence and find the large, non-overlapping open reading frames using the E.coli genetic code and the sites for all Type II and commercially available Type III restriction enzymes that cut the sequence just once.

Suggested NEBuffers for Double Digestion.

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Double digestion (digesting DNA with two restriction enzymes simultaneously) is frequently performed to save time. Our restriction enzymes include universal buffers (refer to the restriction enzyme buffer activity page for relative activity in each buffer), but for some double digests, it may be difficult to select a buffer that is suitable for both enzymes.

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NEBCloner provides a nice protocol taking into account buffer, temperature, additive, as well as star activity differences. Let's see how. Open up NEBCloner and select digestion. Next, choose the two enzymes that you would like to digest simultaneously.
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(NEB) as follows: a 5.24 kb fragment obtained by an Eco RV- Spe I-double digest of AH452 (pXL- BacII-FRT_3xP3DsRed_FRT3_loxN-PUbAmCyan-lox2272 ) Dheneb Personeriasm indistinctly.

Let's see how. Open up NEBCloner and select digestion. Next, choose the two enzymes that you would like to digest simultaneously.
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In this video, you will learn how double digests are made even easier with a tool, NEBCloner. NEBCloner provides a nice protocol taking into account buffer, temperature, additive, as well as star activity differences. Let's see how. Open up NEBCloner and select digestion. Next, choose the two enzymes that you would like to digest simultaneously.

Peak DNA digestion without star activity is best accomplished with conventional We recommend using the Double Digest Finder and use the buffer it recommends. For example, you have old buffer NEBuffer 1 with one of the enzymes and CutSmart Buffer with the second enzyme. If Double Digest Finder recommends to use NEBuffer 1.1, then you can use NEBuffer 1 + BSA for the double digest. Use NEBcloner to find the right products and protocols for each in your traditional cloning workflow, including double digestion buffers. Double digestions can save you time, and this video can offer tips for how to achieve the best results.