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Team:Peking/Notebook/Protocols/DDDP
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[[Team:Peking/Notebook|Notebook]] > [[Team:Peking/Notebook/Protocols|Protocols]] > [[Team:Peking/Notebook/Protocols/DDDP|DNA Double Digestion Protocol]]<html> | [[Team:Peking/Notebook|Notebook]] > [[Team:Peking/Notebook/Protocols|Protocols]] > [[Team:Peking/Notebook/Protocols/DDDP|DNA Double Digestion Protocol]]<html> | ||
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=DNA Double Digestion Protocol= | =DNA Double Digestion Protocol= | ||
==Materials:== | ==Materials:== | ||
| - | + | *DNA sample(s) in water or TE buffer | |
| - | + | ||
| - | + | *10x digestion buffer | |
| - | + | ||
| - | + | *Restriction enzymes (EcoRI or SpeI or XbaI or PstI) | |
| + | |||
| + | *DNA loading buffer (if electrophoresis is subsequent) | ||
| + | |||
| + | *Agarose gel 1.5% (or different depending on expected band sizes) | ||
==Procedure:== | ==Procedure:== | ||
1. Test the concentration of the DNA sample(s). | 1. Test the concentration of the DNA sample(s). | ||
| + | |||
2. Pipet the following into a microfuge tube: | 2. Pipet the following into a microfuge tube: | ||
| - | 20uL reaction system | + | |
| - | DNA around 1ug | + | 20uL reaction system 50uL reaction system |
| - | 10x Digestion buffer 2uL | + | |
| - | 1st Enzyme 1-1.5uL | + | DNA around 1ug around 2.5ug |
| - | 2ndEnzyme 1-1.5uL | + | |
| + | 10x Digestion buffer 2uL 5uL | ||
| + | |||
| + | 1st Enzyme 1-1.5uL 2.5-4uL | ||
| + | |||
| + | 2ndEnzyme 1-1.5uL 2.5-4uL | ||
| + | |||
ddWater Rest of volume Rest of volume | ddWater Rest of volume Rest of volume | ||
| + | |||
3. Incubate at recommended temperature (37.0 degrees) for 2 or 4 hours (0.5~2hfor enzymes of NEB, 4h for enzymes of Takara). | 3. Incubate at recommended temperature (37.0 degrees) for 2 or 4 hours (0.5~2hfor enzymes of NEB, 4h for enzymes of Takara). | ||
| + | |||
4. Take 2 to 5 uLof the digested sample, add loading buffer, and run it on the | 4. Take 2 to 5 uLof the digested sample, add loading buffer, and run it on the | ||
agarose gel to check the result, or take the entire sample to run to extract a | agarose gel to check the result, or take the entire sample to run to extract a | ||
wanted fragment). | wanted fragment). | ||
| + | |||
==Tips:== | ==Tips:== | ||
1. DNA: | 1. DNA: | ||
| - | + | ||
| - | mini prep) | + | *For identification of DNA, use 0.4 ug/uL DNA; (or 2uL from a nice DNA mini prep) |
| - | + | ||
| + | *For cloning, 1ug/uL DNA is enough. | ||
| + | |||
2. Buffer: we’d better use the buffer that comes with the enzyme, which | 2. Buffer: we’d better use the buffer that comes with the enzyme, which | ||
means buffers from other company may cause some abnormal results. | means buffers from other company may cause some abnormal results. | ||
| + | |||
3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the | 3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the | ||
total reaction volume (example: 2 uL for 20 uL reaction system). If you want to do overnight digestion, add less enzyme (example: 1 uL for 20 uL reaction | total reaction volume (example: 2 uL for 20 uL reaction system). If you want to do overnight digestion, add less enzyme (example: 1 uL for 20 uL reaction | ||
system). It is necessary to point that too many enzymes will reduce the efficiency of enzyme digestion with glycerol in it. | system). It is necessary to point that too many enzymes will reduce the efficiency of enzyme digestion with glycerol in it. | ||
| + | |||
4. Gel: make sure to run the uncut DNA as a control along with the digested | 4. Gel: make sure to run the uncut DNA as a control along with the digested | ||
DNA sample(s). And, always run a DNA marker! | DNA sample(s). And, always run a DNA marker! | ||
Latest revision as of 16:36, 12 October 2010
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Contents |
DNA Double Digestion Protocol
Materials:
- DNA sample(s) in water or TE buffer
- 10x digestion buffer
- Restriction enzymes (EcoRI or SpeI or XbaI or PstI)
- DNA loading buffer (if electrophoresis is subsequent)
- Agarose gel 1.5% (or different depending on expected band sizes)
Procedure:
1. Test the concentration of the DNA sample(s).
2. Pipet the following into a microfuge tube:
20uL reaction system 50uL reaction system
DNA around 1ug around 2.5ug
10x Digestion buffer 2uL 5uL
1st Enzyme 1-1.5uL 2.5-4uL
2ndEnzyme 1-1.5uL 2.5-4uL
ddWater Rest of volume Rest of volume
3. Incubate at recommended temperature (37.0 degrees) for 2 or 4 hours (0.5~2hfor enzymes of NEB, 4h for enzymes of Takara).
4. Take 2 to 5 uLof the digested sample, add loading buffer, and run it on the agarose gel to check the result, or take the entire sample to run to extract a wanted fragment).
Tips:
1. DNA:
- For identification of DNA, use 0.4 ug/uL DNA; (or 2uL from a nice DNA mini prep)
- For cloning, 1ug/uL DNA is enough.
2. Buffer: we’d better use the buffer that comes with the enzyme, which means buffers from other company may cause some abnormal results.
3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the total reaction volume (example: 2 uL for 20 uL reaction system). If you want to do overnight digestion, add less enzyme (example: 1 uL for 20 uL reaction system). It is necessary to point that too many enzymes will reduce the efficiency of enzyme digestion with glycerol in it.
4. Gel: make sure to run the uncut DNA as a control along with the digested DNA sample(s). And, always run a DNA marker!
References:
- Current protocols in molecular biology (3.1.1-3.1.2)
