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Verify your Plasmid by Sequencing:.

Optimal Temperature. For digests with >1 µg of DNA used for cloning, it is recommended that you digest for at least 4 hours.

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Digest your DNA: Set up restriction digests for your PCR product and recipient plasmid.

If transforming a selectable marker such as antibiotic resistance, plate the appropriate amount of transformed cells on plates containing the selective agent, grow overnight at 37 degrees (or the appropriate temperature for your plasmid, if other) and proceed to Step 7. . .

It is also critical that as much of the recipient plasmid as possible be cut with both enzymes.

4. After the incubation, place the samples in the refrigerator until the next laboratory period, or proceed directly to step 2 of Lesson 2. Select restriction enzymes to digest your plasmid.

. Overnight digestion without star activity.

restriction enzymes must be mixed gently to avoid inactivating the enzyme.

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Here are solutions to help you prevent and address this issue. Digest your DNA: Set up restriction digests for your PCR product and recipient plasmid.

restriction enzymes must be mixed gently to avoid inactivating the enzyme. After the incubation, place the samples in the refrigerator until the next laboratory period, or proceed directly to step 2 of Lesson 2.

Complete digestion in 15 minutes.
Apr 11, 2012 · fc-falcon">For the ligation to be effective, the restriction enzyme digestion must be stopped.
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It is also critical that as much of the recipient plasmid as possible be cut with both enzymes.

Enzymes that require less than 1.

Label your tube(s). After the incubation, place the samples in the refrigerator until the next laboratory period, or proceed directly to step 2 of Lesson 2. A specific protocol for single digestion using this restriction enzyme can be accessed using our free online tool, NEBcloner.

<span class=" fc-falcon">overnight at room temperature. . Ligations can be done at room temperature or cooler (think 12-16°C. Procedure. class=" fc-falcon">3.

You should see two bands, one the size of your vector and one the size of your new insert.

These enzymes will digest 1 µg of substrate DNA in 5-15 minutes using 1. .

Quick Guide For Restriction Digestion and Analysis DNA RB Ice L P EH Tap Centrifuge Water bath.

2.

Its OK to set this up at room temp.

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