IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/06/21

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The restriction digests were retrieved from the freezer while a gel was set up. After the gel ''fig 2'' had ran for 2 hours, the bands of DNA were visible under ultra violet light so the digest had worked. This meant we could go ahead and remove the fragments of interest, one from each top and middle band of Samples 1 amd 2, one from each middle band of Samples 3 and 4 and one fragment removed from the lower band of Sample 5. We could then isolate the DNA using a gel cleanup system yielding several samples including four large fragments and two extra large fragments of J04450 plasmid DNA and one small fragment of antGPNeo plasmid DNA.  
The restriction digests were retrieved from the freezer while a gel was set up. After the gel ''fig 2'' had ran for 2 hours, the bands of DNA were visible under ultra violet light so the digest had worked. This meant we could go ahead and remove the fragments of interest, one from each top and middle band of Samples 1 amd 2, one from each middle band of Samples 3 and 4 and one fragment removed from the lower band of Sample 5. We could then isolate the DNA using a gel cleanup system yielding several samples including four large fragments and two extra large fragments of J04450 plasmid DNA and one small fragment of antGPNeo plasmid DNA.  
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The next stage involved ligation reactions of various different combinations of J04450 and AntgP-Neo fragments using the isolated DNA fragments from both today and 19th June. The small antGPNeo fragments were ligated with both the large and extra large PSB1C3 fragments seperately. A negative and postive control were set up. For the postive the small fragment and large fragment of PSB1C3 were ligated. The negative control involved single fragments of antGPNeo and J04450 to see if they self ligate. The samples were left to incubate at 4°C overnight.   
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The next stage involved ligation reactions of various different combinations of J04450 and AntgP-Neo fragments using the isolated DNA fragments from both today and 19th June. The small antGPNeo fragments were ligated with both the large and extra large J04450 fragments seperately. A negative and postive control were set up. For the postive the small fragment and large fragment of J04450 were ligated. The negative control involved single fragments of antGPNeo and J04450 to see if they self ligate. The samples were left to incubate at 4°C overnight.   
  [[Image:800px-21 june.jpg|right|thumb|Fig 2: Third analysis of restriction digest products by agarose gel electrophoresis, J04450 (Samples 1-4) and AntGPneo plasmids (Sample 5) cut with EcoR1 and Pst1. ]]
  [[Image:800px-21 june.jpg|right|thumb|Fig 2: Third analysis of restriction digest products by agarose gel electrophoresis, J04450 (Samples 1-4) and AntGPneo plasmids (Sample 5) cut with EcoR1 and Pst1. ]]

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The restriction digests were retrieved from the freezer while a gel was set up. After the gel fig 2 had ran for 2 hours, the bands of DNA were visible under ultra violet light so the digest had worked. This meant we could go ahead and remove the fragments of interest, one from each top and middle band of Samples 1 amd 2, one from each middle band of Samples 3 and 4 and one fragment removed from the lower band of Sample 5. We could then isolate the DNA using a gel cleanup system yielding several samples including four large fragments and two extra large fragments of J04450 plasmid DNA and one small fragment of antGPNeo plasmid DNA. The next stage involved ligation reactions of various different combinations of J04450 and AntgP-Neo fragments using the isolated DNA fragments from both today and 19th June. The small antGPNeo fragments were ligated with both the large and extra large J04450 fragments seperately. A negative and postive control were set up. For the postive the small fragment and large fragment of J04450 were ligated. The negative control involved single fragments of antGPNeo and J04450 to see if they self ligate. The samples were left to incubate at 4°C overnight.

Fig 2: Third analysis of restriction digest products by agarose gel electrophoresis, J04450 (Samples 1-4) and AntGPneo plasmids (Sample 5) cut with EcoR1 and Pst1.
Fig 2: Third analysis of restriction digest products by agarose gel electrophoresis, J04450 (Samples 1-4) and AntGPneo plasmids (Sample 5) cut with EcoR1 and Pst1.


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