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Team:Brown/Notebook Protocols/redigest
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(New page: {{Brown}} DNA digestion protocol & hints ---- Overview: Although it is pretty standard to digest DNA with restriction enzymes, here are a standardized protocol and some hints...) |
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| - | DNA digestion protocol & hints | + | '''<big>DNA digestion protocol & hints</big>''' |
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| + | Overview: Although it is pretty standard to digest DNA with restriction enzymes, here is a standardized protocol and some hints… | ||
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Materials: | Materials: | ||
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• DNA sample in water or TE buffer | • DNA sample in water or TE buffer | ||
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• 10x digestion buffer | • 10x digestion buffer | ||
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• restriction enzyme | • restriction enzyme | ||
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• DNA loading buffer | • DNA loading buffer | ||
• Agarose gel 0.8% (or different depending on expected band sizes) | • Agarose gel 0.8% (or different depending on expected band sizes) | ||
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Procedure: | Procedure: | ||
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1. Pipet the following into a microfuge tube: | 1. Pipet the following into a microfuge tube: | ||
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20 µl reaction 50 µl reaction | 20 µl reaction 50 µl reaction | ||
DNA 0.1 to 4 µg 0.1 to 4 µg | DNA 0.1 to 4 µg 0.1 to 4 µg | ||
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buffer 2 | buffer 2 | ||
µl 5 µl | µl 5 µl | ||
| - | Enzyme | + | Enzyme (as appropriate) |
| - | Water Rest of volume | + | Water (Rest of volume) |
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2. Add the enzyme (1-5u/µg DNA) | 2. Add the enzyme (1-5u/µg DNA) | ||
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3. Incubate at recommended temperature for an hour. | 3. Incubate at recommended temperature for an hour. | ||
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4. Take 2 to 5 µl of the digested sample, add loading buffer, and run on agarose gel to | 4. Take 2 to 5 µl of the digested sample, add loading buffer, and run on agarose gel to | ||
check the result. | check the result. | ||
Tips: | Tips: | ||
| - | 1. DNA: | + | |
| + | 1. | ||
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| + | DNA: | ||
• for checking DNA, use 0.1 µg; (or 4-8 µl from a good DNA mini prep) | • for checking DNA, use 0.1 µg; (or 4-8 µl from a good DNA mini prep) | ||
| - | • For cloning, 4 µg DNA is enough | + | • For cloning, 4 µg DNA is enough |
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2. Buffer: besides the buffer that comes with the enzyme, buffers from other company | 2. Buffer: besides the buffer that comes with the enzyme, buffers from other company | ||
can be used, too (as long as the contents are the same) | can be used, too (as long as the contents are the same) | ||
| + | |||
3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the total | 3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the total | ||
reaction volume (example: 2 µl for 10 µl reaction) | reaction volume (example: 2 µl for 10 µl reaction) | ||
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4. Incubation time: can be longer than 1 hr, but don’t do overnight digestion if you don’t | 4. Incubation time: can be longer than 1 hr, but don’t do overnight digestion if you don’t | ||
| - | have to (genomic DNA requires overnight digestion) | + | have to (genomic DNA requires overnight digestion) |
| - | + | ||
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| - | + | 5. Gel: make sure to run the uncut DNA along with the digested DNA. And, always run a DNA marker! | |
| - | + | ---- | |
| + | References: ''Current protocols in molecular biology'' (3.1.1 - 3.1.2) | ||
Revision as of 00:21, 22 October 2009
DNA digestion protocol & hints
Overview: Although it is pretty standard to digest DNA with restriction enzymes, here is a standardized protocol and some hints…
Materials:
• DNA sample in water or TE buffer
• 10x digestion buffer
• restriction enzyme
• DNA loading buffer
• Agarose gel 0.8% (or different depending on expected band sizes)
Procedure:
1. Pipet the following into a microfuge tube:
20 µl reaction 50 µl reaction DNA 0.1 to 4 µg 0.1 to 4 µg 10x Digestion buffer 2 µl 5 µl Enzyme (as appropriate) Water (Rest of volume)
2. Add the enzyme (1-5u/µg DNA)
3. Incubate at recommended temperature for an hour.
4. Take 2 to 5 µl of the digested sample, add loading buffer, and run on agarose gel to check the result.
Tips:
1.
DNA: • for checking DNA, use 0.1 µg; (or 4-8 µl from a good DNA mini prep) • For cloning, 4 µg DNA is enough
2. Buffer: besides the buffer that comes with the enzyme, buffers from other company can be used, too (as long as the contents are the same)
3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the total reaction volume (example: 2 µl for 10 µl reaction)
4. Incubation time: can be longer than 1 hr, but don’t do overnight digestion if you don’t have to (genomic DNA requires overnight digestion)
5. Gel: make sure to run the uncut DNA along with the digested DNA. And, always run a DNA marker!
References: Current protocols in molecular biology (3.1.1 - 3.1.2)
