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

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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 wrong 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


Image:Dig_HydA_hydG_pSB1C3.jpg

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, apparently this happened because only one enzime worked, which is strange because the reactions took place under the same conditions that HydG digestions. Besides Helena is tring to take off the tag (I will probably continue with her work latter).

The digestion for hydG were well. At the beginnig we were wondering why there were two marks near 1700bp (which is HydG's size) and another small band in the lane. Then we discovered that the plasmid in which the people from Mr. gene sent us the part has an internal restriction site for PstI. The fragment's size is very similar to HydG's size, the difference is that the fragment is flanked by two Pst sites so it will not be correctly ligated to the plasmid. If both fragments ligate together we will see it in the size.

Nevertheless because of all these problems Pablo will repeat the digestion but this time with EcoRI and Spe.


HydG extraction

Abstract

I need to do a band extraction to recover the HydG's double digestion. I ran a LMP gel and I used the QIAGEN™ QIAquick Gel Extraction Kit to extract the band near 1700 bp.

Details

I fused the sample 1-2 and the samples 3-4 so I only had two lanes in the gel, then to do the extraction I fused everything in a single sample. The marks in the gel were very diffuse so I cut out all the part that was shinning the most near 1700bp.

Gel details

  • 18μL template + 1.8μL dye 10X
  • 4.5μL ladder 250bp + 0.5μL dye
  • 83V, 100min

Extraction details

I recovered 60mg from the gel and then I followed IAGEN™ QIAquick Gel Extraction Kit protocol. At the end I washed with 40μL of EB buffer after 1 minute of repose to have more concentration.



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