Team:Amsterdam/Notebook/Protocols/Digestion

From 2011.igem.org

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{{:Team:Amsterdam/Header}}
{{:Team:Amsterdam/Header}}
-
==Restriction Digests==
+
=Digestion: Making new constructs=
-
At iGEM HQ we use this protocol for restriction digests along with enzymes purchased from NEB.
+
We used this protocol for digesting the biobricks.
-
 
+
<br><br>
-
===Materials===  
+
==Materials==  
-
*PCR tube
+
*PCR tubes
*dH20
*dH20
*Enzymes (EcoRI, XbaI, SpeI, PstI)
*Enzymes (EcoRI, XbaI, SpeI, PstI)
*BSA
*BSA
-
*Enzyme Buffer (NEBuffer 2)*
+
*Enzyme Buffer (NEBuffer 2)
 +
 
 +
<i>Note: All materials should be kept on ice during preparation.</i>
 +
<br><br>
 +
 
 +
==Protocol==
 +
 
 +
{| border="1"
 +
!align="left"|Vector
 +
!align="right"|μl
 +
|-
 +
|DNA
 +
|align="right"|10
 +
|-
 +
|NEB buffer 2
 +
|align="right"|2
 +
|-
 +
|BSA
 +
|align="right"|0,5
 +
|-
 +
|SpeI
 +
|align="right"|1
 +
|-
 +
|PstI
 +
|align="right"|1
 +
|-
 +
|H2O
 +
|align="right"|5,5
 +
|- style="font-style:italic;"
 +
|Total
 +
|align="right"|20 μl
 +
|}
 +
<br/>
 +
{| border="1"
 +
!align="left"|Insert
 +
!align="right"|μl
 +
|-
 +
|DNA
 +
|align="right"|10
 +
|-
 +
|NEB buffer 2
 +
|align="right"|2
 +
|-
 +
|BSA
 +
|align="right"|0,5
 +
|-
 +
|XbaI
 +
|align="right"|0,5
 +
|-
 +
|PstI
 +
|align="right"|1
 +
|-
 +
|H2O
 +
|align="right"|6
 +
|- style="font-style:italic;"
 +
|Total
 +
|align="right"|20 μl
 +
|}
 +
 
 +
 
 +
1. Incubate the restriction digest at 37&deg;C for 2 hours.<br>
 +
2. Incubate at 80&deg;C for 20 min to inactivate enzymes.<br>
 +
3. Store at -4&deg;C.
 +
 
 +
<br><br>
 +
 
 +
==Double digest==
 +
To check if the enzymes digest the plasmid as expected, the following digestion should be performed:<br>
 +
1. First use only SpeI(vector) and XbaI(insert).<br>
 +
2. Incubate at 37&deg;C for 1 hour.<br>
 +
3. take 1 μl of each sample.<br>
 +
4. add PstI to all the samples.<br>
 +
5. Incubate at 37&deg;C for 1 hour.<br>
 +
6. take 1 μl of each sample.<br>
 +
7. take 1 μl of each undigested sample.<br>
 +
8. fill each of the three samples up to 10 μl including LD and check them on agarose gel.<br>
 +
<br><br>
 +
=Digestion: Changing the backbone=
 +
We used this protocol to transfer the construct in a new backbone.
 +
<br><br>
 +
==Materials==
 +
*PCR tubes
 +
*dH20
 +
*Enzymes (EcoRI, PstI)
 +
*BSA
 +
*Enzyme Buffer (NEBuffer 2)
 +
<i>Note: All materials should be held on ice during preparation.</i>
 +
<br><br>
 +
==Protocol==
 +
 
 +
{| border="1"
 +
!align="left"|Sample
 +
!align="right"|μl
 +
|-
 +
|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;"
 +
|Total
 +
|align="right"|20 μl
 +
|}
 +
<br/>
 +
 
 +
 
 +
1. Incubate the restriction digest at 37&deg;C for 2 hours.<br>
 +
2. Incubate at 80&deg;C for 20 min to heat kill the enzymes.<br>
 +
3. Store at -4&deg;C.
 +
 
 +
<br><br>
 +
==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&deg;C for 1 hour.<br>
 +
3. take 1 μl of each sample.<br>
 +
4. add PstI to all the samples.<br>
 +
5. Incubate at 37&deg;C for 1 hour.<br>
 +
6. take 1 μl of each sample.<br>
 +
7. take 1 μl of each undigested sample.<br>
 +
8. fill each of the three samples up to 10 μl including LD and check them on agarose gel.<br>
-
Notes: You should keep all materials on ice.
+
=Purification=
 +
Samples are often purified after digestion using a gel extraction kit. Gel extraction protocols and kits from [http://www.qiagen.com/hb/qiaquickgelextractionkit_en Qiagen] and [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0691.pdf Fermentas] were used.<br><br>
-
===Protocol===
 
-
1. Add 500ng of DNA to be digested, and adjust with dH20 for a total volume of 42.5ul.<br>
 
-
2. Add 5ul of NEBuffer 2 to the tube.<br>
 
-
3. Add 0.5ul of BSA to the tube.<br>
 
-
4. Add 1ul of your first enzyme.<br>
 
-
5. Add 1ul of your second enzyme.<br>
 
-
6. There should be a total volume of 50ul. Mix well and spin down.<br>
 
-
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>
 
-
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}}

Latest revision as of 23:59, 21 September 2011

Contents

Digestion: Making new constructs

We used this protocol for digesting the biobricks.

Materials

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

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

Protocol

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


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


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



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°C for 1 hour.
3. take 1 μl of each sample.
4. add PstI to all the samples.
5. Incubate at 37°C for 1 hour.
6. take 1 μl of each sample.
7. take 1 μl of each undigested sample.
8. fill each of the three samples up to 10 μl 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 μl
DNA 10
NEB buffer 2 2
BSA 0,5
EcoRI 0,5
PstI 1
H2O 6
Total 20 μl



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



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°C for 1 hour.
3. take 1 μl of each sample.
4. add PstI to all the samples.
5. Incubate at 37°C for 1 hour.
6. take 1 μl of each sample.
7. take 1 μl of each undigested sample.
8. fill each of the three samples up to 10 μl including LD and check them on agarose gel.

Purification

Samples are often purified after digestion using a gel extraction kit. Gel extraction protocols and kits from [http://www.qiagen.com/hb/qiaquickgelextractionkit_en Qiagen] and [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0691.pdf Fermentas] were used.

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