dsap Flashcards
step 1
Downloading the Sequence - download waveform software (4peaks/FinchTV), download For Sequence and open file
step 2
Cropping Ends - is sequence readable
which base does unedited cDNA insert start
first base that’s not G or C after GGCCGG
which base does the unedited cDNA insert end
after the polyA tail, no polyAtail –> choose last readable base
poor quality can be caused by…
1) ethanol in plasmid minipreps (forget to do second spin in wash step)
2) forgetting to do wash step entirely –> SDS eluted with DNA
3) mixed templates (2 plasmids present)
base colors
A - Green
T - Red
C - Blue
G - Black
Blast Purpose
determine evolutionary relationship with other organisms to understand gene function
BlastN
Nucleotide –> Nucleotide
expect value: closer it is to 0 –> more likely the match is meaningful
BlastX
nucleotide –> protein
determine if our seuqnece likely codes for a protein
BlastP
protein –> protein
do this when you know that your sequence codes for a protein from BlastX (might code for another functional molecule)
what do you need to fill out the table
accession #, definition, organism, query start, query end, e value
potential reasons for mismatches
1) degeneracy of amino acids (multiple triplets code for same amino acid)
2) discontinuous alignment
3) small gaps (frame -shifts)
4) gaps in query & subject sequences
what is a good identity percentage
greater than 30%
what does graphic alignment summary show
contiguous matching regions
black: < 40 bps
blue: 40 - 50 bps
green: 50 - 80 bps
pink: 80 - 200 bps
red: >200 bps
best reading frame
M at the Start, multiple N bases, PolyA tail at 3’ end
