Restricting Plasmids (Double Restriction)

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{{:Team:Newcastle University/Template:UnderTheProtocol|page-title=[[Restricting Plasmids (Double Restriction)]]}}
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[[Image:Jess1.jpg|thumb|right|200px| Jess enjoying pipetting buffer into her restriction mixture.]]
[[Image:Jess1.jpg|thumb|right|200px| Jess enjoying pipetting buffer into her restriction mixture.]]
We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.
We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.
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Total volume = 30μl
Total volume = 30μl
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[[Image:DSC01597.jpg|thumb|left|200px|The DNA purification kit we use to purify enzymatic reaction mixtures.]]
 
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* Incubate solutions for 90 minutes in a  37°C water bath.
 
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* If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.
 
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[[Image:DSC01597.jpg|200px]]
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The DNA purification kit we use to purify enzymatic reaction mixtures.
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* Incubate solutions for 90 minutes in a  37°C water bath.
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* If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.
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'''Back to [[Team:Newcastle University/Protocols]]'''
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'''Back to [[Team:Newcastle University/Notebook]]'''
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Latest revision as of 16:01, 28 October 2008

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Home >> Wet Lab >> Protocols >> Restricting Plasmids (Double Restriction)

Jess enjoying pipetting buffer into her restriction mixture.

We have conducted restrictions in varying concentrations and total volumes; however they all follow the same basic procedure. Below are two of the different restrictions we carried out.


  • 46μl MillQ H2O
  • 10μl 10 x buffer
  • 40μl plasmid sample
  • 2μl enzyme 1
  • 2μl enzyme 2

Total volume = 100μl

Concentrated

  • 10μl MilliQ H2O
  • 3μl 10 X buffer
  • 10μl plasmid sample
  • 1μl enzyme 1
  • 1μl enzyme 2

Total volume = 30μl


DSC01597.jpg

The DNA purification kit we use to purify enzymatic reaction mixtures.

  • Incubate solutions for 90 minutes in a 37°C water bath.
  • If DNA is to be run on a gel and a band cut out, this can be done without purifying the DNA. For all other downstream applications or for storage, solution must be purified to remove the enzymes.