Are there any errors (mismatches, gaps, insertions) when you check the alignment against the expected sequences?

You should have, by now, determined the exact sequence of the polynucleotide inserts that you transformed into the vectors in Week 10. Hint: Determine the exact sequence of PCR products from the templates and primers used in Week 6; then determine which nucleotides were removed from these by the restriction enzyme digests performed in Week 8 and Week 9. The table in the file Summary of Results summarises the quality of the reads for each sequencing reaction; the key explaining the columns is at the bottom of the table. Find your data to determine if you have any quality sequencing results. Each pair has four entries in the table, one for each sequencing reaction you set up in Week 10. Pair 10 have eight entries, four labelled 101 and four labelled 102 – this numbering should make sense to you. A quick look by me and I think Pairs 1, 5 and 17 do not have any reads to analyse – please use the data from a pair working in the same bay – state which pair in your write-up. Each of the folders below contains one file for each reaction (72 files in each folder): Results Sequencing Chromatograms open in Finchtv (on PC s in lab in Peter Wilson building) Nucleotide Sequences Open and analyse each of the pdf files of your Results; these contain a summary of the results for that sequencing reaction with the following sections General Information – the running of sequencing experiment Quality Graph – shows extent of the quality of the data through the read Sequence – shows what sequence has been identified by the read Chromatogram Trace – is what is says
You should now know which of your sequencing reactions has generated a quality read of a nucleotide sequence. Validate these sequence results using the same protocol you followed in Week 5, Part 10. You can use any sequences identified by copying from the summary in the Results folder, using the fasta sequence from the Nucleotide Sequences folder, or extracting the sequence from the chromatogram as you did in Week 5.
For your write-up, you need to consider the following: Did you get complete reads, covering 100 % of your target sequence, from beginning to end, in either direction forward or reverse, with just one primer? If not, what is the coverage (report as % of the total number of expected nucleotides)? If you have reads with both primers for one construct, do you get 100 % coverage and, if not, what is the coverage? Are there any errors (mismatches, gaps, insertions) when you check the alignment against the expected sequences? If yes, check the chromatogram to see if the software has misinterpreted the result or if it is a genuine error in the sequence. You will need to use Finchtv to view the chromatogram, on the PCs in the PC lab in the Peter Wilson building.

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