Dahlquist:Analyzing Yeast Deletion Strain Barcode Sequences

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This page is for writing out the protocol for Analyzing Yeast Deletion Strain Barcode Sequences.

Step 1

manually go through sequence, matching nucleotides to corresponding peaks and making necessary corrections
search sequence for N's, replacing them with correct nucleotide if highest peak can be determined
if N's at the beginning and end of sequence cannot correctly be identified and matched to the correct peak, cut out that portion of the sequence (be sure to only cut out beginning and end of sequence, DO NOT cut N's in the middle of sequence)
edit computer text file version of sequence, cutting out beginning/ end portions and replacing N's with correct nucleotides
- Note: When opening text files begin by unzipping folder and extracting files. Next open notepad app on computer. Once notepad is open click file open and navigate to downloaded file to open text version of sequences.

go to Dahlquist Protocols page
right-click on the link for the List of Yeast Deletion Project Primers and then "Save link as" to download it. If you just left-click, it will open in the browser because it is a text file. Once you've downloaded it, you can open the file in Excel. You will need to know the systematic name for the genes you are using, which you can look up on the Saccharomyces Genome Database (SGD).
locate systematic name in excel sheet

now run a Blast 2 sequence match https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq
insert corrected/edited gene sequence in top box
insert corresponding tag sequence of the gene (uptag or down tag), which is found in the excel spread sheet, into the second box
Use following primer key to determine whether uptag or down tag sequence must be inserted
Key:
- A-KanB (uptag)
- KanC-D (downtag)
- A-D (A = uptag & D= downtag)
NOTE: when inserting down tag into Blast with corresponding corrected gene sequence, be sure to use the reverse gene complement, which can be obtained by entering the sequence into this website http://www.bioinformatics.org/sms/rev_comp.html