Students,
To prepare the reports from the Southern hybridization experiment (TP3) each group should consider the following results:
1) agarose gel in which you have run your PCR reaction;
2) agarose gel referring to genomic DNA digestion;
3) agarose gel referring to the result obtained after purification of the probe (“Purified PCR products “)
4) hybridization membrane
Here you will find all the files you need. The images of the membrane will be uploaded as soon as A5 is completed.
In silico analysis
In order for you to be able to match the results obtained in the membrane with those that you were expecting to obtain, you could try to make the restriction map of the genomic region that includes the pgmG gene. To do this you should:
1. Access the NCBI page dedicated to the genome of S. elodea
(http://www.ncbi.nlm.nih.gov/genome/?term=sphingomonas+elodea)
2. Obtain the nucleotide sequence of the pgmG gene
https://www.ncbi.nlm.nih.gov/nuccore/AF167367.1?&feature=CDS#feature_6103618_CDS_0
3. Click Tools and then the BLAST Genome option.
4. Past to the BLAST screen the nucleotide sequence of the pgmG gene. Make sure that you untick the Complete Genome option and instead tick the draft genome option
5. The result obtained refers to the genomic region where the pgmG gene is located. This gene is inserted between positions 15,985 and 17,546. In the alignment click "Graphics”
6. To perform the restriction analysis, choose a fragment containing 15kb upstream and 15 kb downstream from the region of pgmG. To do that click on Tools and then Go to – insert the coordinates of interest. Extract the nucleotide sequence by clicking Tools, Download FASTA visible range.
7. You will be able to use this DNA sequence in any tool you chose that makes suitable restriction maps (for example NEB Cutter). In NEB Cutter use the option “Custom Digest” to select the enzymes of interest and then ”View gel” to check the size of the restriction fragments. You should then evaluate in which fragment the region of pgmG is present.
In case you miss some data and/or have doubts to clarify please feel free to contact me by email.
Filipa Mendes
