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.]]
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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[[Image:Jess1.jpg|thumb|right|300px| Jess enjoying pipetting buffer into her restriction mixture.]]
 
* 46μl MillQ H2O
* 46μl MillQ H2O
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* 2μl enzyme 2
* 2μl enzyme 2
Total volume = 100μl
Total volume = 100μl
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'''Concentrated'''
'''Concentrated'''
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* 1μl enzyme 2
* 1μl enzyme 2
Total volume = 30μl
Total volume = 30μl
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[[Image:DSC01597.jpg|200px]]
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The DNA purification kit we use to purify enzymatic reaction mixtures.
* Incubate solutions for 90 minutes in a  37°C water bath.
* 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.
* 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.

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.