Team:Amsterdam/Notebook/Protocols/Digestion

From 2011.igem.org

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{{:Team:Amsterdam/Header}}
{{:Team:Amsterdam/Header}}
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==Restriction Digests==
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=Digestion=
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At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.
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We used this protocol for the digestion of the biobricks.
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===Materials===  
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==Materials==  
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*PCR tube
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*PCR tubes
*dH20
*dH20
*Enzymes (EcoRI, XbaI, SpeI, PstI)
*Enzymes (EcoRI, XbaI, SpeI, PstI)
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*Enzyme Buffer (NEBuffer 2)*
*Enzyme Buffer (NEBuffer 2)*
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Notes: You should keep all materials on ice.
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Note: All materials should be held on ice during preparation.
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==Protocol==
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{| border="1"
 +
!align="left"|Vector
 +
!align="right"|ul
 +
|-
 +
|DNA
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|align="right"|10
 +
|-
 +
|NEB buffer 2
 +
|align="right"|2
 +
|-
 +
|BSA
 +
|align="right"|0,5
 +
|-
 +
|SpeI
 +
|align="right"|1
 +
|-
 +
|PstI
 +
|align="right"|1
 +
|-
 +
|H2O
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|align="right"|5,5
 +
|- style="font-style:italic;"
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|Total
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|align="right"|20 ul
 +
|}
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<br/>
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{| border="1"
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!align="left"|Insert
 +
!align="right"|ul
 +
|-
 +
|DNA
 +
|align="right"|10
 +
|-
 +
|NEB buffer 2
 +
|align="right"|2
 +
|-
 +
|BSA
 +
|align="right"|0,5
 +
|-
 +
|XbaI
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|align="right"|0,5
 +
|-
 +
|PstI
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|align="right"|1
 +
|-
 +
|H2O
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|align="right"|6
 +
|- style="font-style:italic;"
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|Total
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|align="right"|20 ul
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|}
 +
 
 +
 
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1. Incubate the restriction digest at 37C for 2 hours.<br>
 +
2. Incubate at 80C for 20min to heat kill the enzymes.<br>
 +
3. Store at -4C.
 +
 
 +
 
 +
 
 +
 
 +
==Double digest==
 +
To check if the enzymes digest the plasmid as expected, the following digestion should be performed:<br>
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1. First use only SpeI(vector) and XbaI(insert).<br>
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2. Incubate at 37 degrees Celsius for 1 hour.<br>
 +
3. take 1 ul of each sample.<br>
 +
4. add PstI to all the samples.<br>
 +
5. Incubate at 37 degrees Celsius for 1 hour.<br>
 +
6. take 1 ul of each sample.<br>
 +
7. take 1 ul of each undigested sample.<br>
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8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.<br>
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===Protocol===
 
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1. Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.<br>
 
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2. Add 5ul of NEBuffer 2 to the tube.<br>
 
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3. Add 0.5ul of BSA to the tube.<br>
 
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4. Add 1ul of your first enzyme.<br>
 
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5. Add 1ul of your second enzyme.<br>
 
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6. There should be a total volume of 50ul. Mix well and spin down.<br>
 
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7. Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes.<br> ''We incubate in a thermocycler with a heated lid''<br>
 
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8. Run a portion of the digest on a gel, to check that both plasmid and part length are accurate. You may also use 2ul of the digest (20ng of DNA) for ligations.
 
{{:Team:Amsterdam/Footer}}
{{:Team:Amsterdam/Footer}}

Revision as of 13:24, 13 September 2011

Contents

Digestion

We used this protocol for the digestion of the biobricks.

Materials

  • PCR tubes
  • dH20
  • Enzymes (EcoRI, XbaI, SpeI, PstI)
  • BSA
  • Enzyme Buffer (NEBuffer 2)*

Note: All materials should be held on ice during preparation.

Protocol

Vector ul
DNA 10
NEB buffer 2 2
BSA 0,5
SpeI 1
PstI 1
H2O 5,5
Total 20 ul


Insert ul
DNA 10
NEB buffer 2 2
BSA 0,5
XbaI 0,5
PstI 1
H2O 6
Total 20 ul


1. Incubate the restriction digest at 37C for 2 hours.
2. Incubate at 80C for 20min to heat kill the enzymes.
3. Store at -4C.



Double digest

To check if the enzymes digest the plasmid as expected, the following digestion should be performed:
1. First use only SpeI(vector) and XbaI(insert).
2. Incubate at 37 degrees Celsius for 1 hour.
3. take 1 ul of each sample.
4. add PstI to all the samples.
5. Incubate at 37 degrees Celsius for 1 hour.
6. take 1 ul of each sample.
7. take 1 ul of each undigested sample.
8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.

Retrieved from "http://2011.igem.org/Team:Amsterdam/Notebook/Protocols/Digestion"