Team:Peking S/lab/protocol

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<font size=6> <font color="#FFFFFF">Protocol</font></FONT>
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{{Template:Http://2011.igem.org/Team:Peking S/protocal}}
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=DNA Double Digestion Protocol=
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<html>
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<a href="https://static.igem.org/mediawiki/2010/8/84/DNA_double_digestion_protocol.pdf"target="blank"><img src="https://static.igem.org/mediawiki/2010/9/91/PKU_Adobe_Reader_Logo.jpg" width=20>download PDF version</a><br>
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</html>
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==Materials:==
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*DNA sample(s) in water or TE buffer
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*10x digestion buffer
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*Restriction enzymes (EcoRI or SpeI or XbaI or PstI)
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*DNA loading buffer (if electrophoresis is subsequent)
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*Agarose gel 1.5% (or different depending on expected band sizes)
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==Procedure:==
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1. Test the concentration of the DNA sample(s).
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2. Pipet the following into a microfuge tube:
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20uL reaction system    &nbsp; &nbsp;                50uL reaction system
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DNA around 1ug      &nbsp; &nbsp;                around 2.5ug
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10x Digestion buffer 2uL  &nbsp; &nbsp;                5uL
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1st Enzyme 1-1.5uL  &nbsp; &nbsp;                2.5-4uL
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2ndEnzyme 1-1.5uL    &nbsp; &nbsp;                  2.5-4uL
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ddWater Rest of volume Rest of volume
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3. Incubate at recommended temperature (37.0 degrees) for 2 or 4 hours (0.5~2hfor enzymes of NEB, 4h for enzymes of Takara).
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4. Take 2 to 5 uLof the digested sample, add loading buffer, and run it on the
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agarose gel to check the result, or take the entire sample to run to extract a
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wanted fragment).
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==Tips:==
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1. DNA:
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*For identification of DNA, use 0.4 ug/uL DNA; (or 2uL from a nice DNA mini prep)
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*For cloning, 1ug/uL DNA is enough.
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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.
 +
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4. Gel: make sure to run the uncut DNA as a control along with the digested
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DNA sample(s). And, always run a DNA marker!
 +
 +
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==References:==
 +
*Current protocols in molecular biology (3.1.1-3.1.2)

Latest revision as of 08:31, 2 October 2011


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Protocol


Double Digestion |Gel Extraction|Miniprep | Chemical Inducible Expression of GFP |DNA Purification |Ligation of Insert DNA | PCR |Preparation of Competent Cells| Site-directed Mutagenesis| Transformation|


DNA Double Digestion Protocol

download PDF version

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)
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