IGEM:UNAM LCG/2009/Notebook/Hydrobium etli/2011/09/23: Difference between revisions

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Line 64: Line 64:
   9. Ladder [http://www.fermentas.com/en/products/all/dna-electrophoresis/ogeneruler-dna-ladders/sm117-ogeneruler-mix fermentas]
   9. Ladder [http://www.fermentas.com/en/products/all/dna-electrophoresis/ogeneruler-dna-ladders/sm117-ogeneruler-mix fermentas]
   10. pSB1C3
   10. pSB1C3
   11-end. empty
   11-rest. empty




Revision as of 18:24, 23 September 2011

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HydA and Hyd G digestion

Abstract

I made a double digestion from HydA and HydG with EcoRI and PstI to extract them from the plasmid in which they were sent to us by Mr. Gene. The objective is to ligate this parts with the plasmid pSB1C3 to send them to the registry.

Apparently something went grong in the digestion of HydA.

Details

I prepared 4 sample for each of the parts (hydA and hydG) so I made 4 digestions for each part but this digestions were replicas, all had the same ammounts of components.

Digestion details

Reactive Ammount (μL)
MiliQ Water 9
Buffer (EcoRI) 2
BSA 10X 2
Template DNA 5
EcoRI (NEB™) 0.5
PstI (NEB™) 0.5
Total 20
  • I incubate the reaction for 3:10 hours at 37°C
  • Then I inactivate the enzymes by incubating at 80°C for 20 minutes.
  • I decided to use that Buffer according to NEB webpage double-digestion guide.

Results

I ran a gel to see if the digestions were well.

  • 120V, 38 min.

Gel image

Lanes: 

 1. HydA 1
 2. HydA 2
 3. HydA 3
 4. HydA 4
 5. HydG 1
 6. HydG 2
 7. HydG 3
 8. HydG 4
 9. Ladder fermentas
 10. pSB1C3
 11-rest. empty


Gel analysis

The double digestion of pSB1C3 was made by Gustavo, He put it in this gel to see the concentration and calculate the ammaunt he will need to do a ligation, I will use this same sample to do the ligation in the next step.

It apperas that HydA digestion did not work out because there is a single band, I have to ask Pae if this is the expected size anyway. Besides Helena is tring to take off the tag.

I am not sure if the diggestions for HydG were well because I do not know what are the expected sizes, Pae will analize it and tell me.


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