Team:Amsterdam/Notebook/Protocols/Digestion

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7. take 1 ul of each undigested 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>
8. fill each of the three samples up to 10 ul including LD and check them on agarose gel.<br>
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Revision as of 19:41, 16 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"