Team:Amsterdam/Notebook/Protocols/Digestion

From 2011.igem.org

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{{:Team:Amsterdam/Header}}
{{:Team:Amsterdam/Header}}
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=Digestion=
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=Digestion: Making new constructs=
We used this protocol for the digestion of the biobricks.
We used this protocol for the digestion of the biobricks.
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2. Incubate at 80C for 20min to heat kill the enzymes.<br>  
2. Incubate at 80C for 20min to heat kill the enzymes.<br>  
3. Store at -4C.
3. Store at -4C.
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8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.<br>
8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.<br>
 +
=Digestion: Changing the backbone=
 +
We used this protocol to transfer the construct in a new backbone.
 +
 +
==Materials==
 +
*PCR tubes
 +
*dH20
 +
*Enzymes (EcoRI, PstI)
 +
*BSA
 +
*Enzyme Buffer (NEBuffer 2)
 +
<br>
 +
 +
Note: All materials should be held on ice during preparation.
 +
 +
==Protocol==
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{| border="1"
 +
!align="left"|sample
 +
!align="right"|ul
 +
|-
 +
|DNA
 +
|align="right"|10
 +
|-
 +
|NEB buffer 2
 +
|align="right"|2
 +
|-
 +
|BSA
 +
|align="right"|0,5
 +
|-
 +
|EcoRI
 +
|align="right"|0,5
 +
|-
 +
|PstI
 +
|align="right"|1
 +
|-
 +
|H2O
 +
|align="right"|6
 +
|- 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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 +
 +
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>
 +
1. First use only EcoRI.<br>
 +
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>
 +
8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.<br>
{{:Team:Amsterdam/Footer}}
{{:Team:Amsterdam/Footer}}

Revision as of 13:38, 13 September 2011

Contents

Digestion: Making new constructs

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.

Digestion: Changing the backbone

We used this protocol to transfer the construct in a new backbone.

Materials

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


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

Protocol

sample ul
DNA 10
NEB buffer 2 2
BSA 0,5
EcoRI 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 EcoRI.
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"