Recipes & Protocols

Tools

• Ensembl
• Primer3Plus
• USCS In-silico PCR
• IDT OligoAnalyzer
• Reverse complement
• GenoBase Keio
• DNA Restriction Site Analyzer

Gene/Protein Info

• NCBI: EF-TU CD

Protocols

• Choosing primers for q-PCR

Primer designing for gene expression profiling (bacteria)

➢Get the gene name (eg: MYD88)

• Go to NCBI (ncbi.nlm.nih.gov), and search in ‘Gene’ for the gene sequence.
• Type in REL606 (that is the E.coli strain we use, unless stated otherwise) together with the gene name, and get the NM number of the most common transcript variant. (eg: NM_002468.4 for MYD88)
• Click on the link and go to the gene description page.
• Scroll down and clink on the link to find the CDS, or the RefSeq.
• Copy the CDS sequence.
• Go to the Primer 3 Plus website (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) and paste the CDS sequence in the search box.
• All the parameters for optimal primers are usually preset. Do not change anything.
• Click “pick primers’.
• When the selection of primers comes up, choose a pair that is closest to the 3’ end.
• Check the primer is 20bp long and the product size is between 150-200bp.

• Blast to check the specificity of the primer pair (http://blast.ncbi.nlm.nih.gov/)
• Paste the forward and reverse primer sequences one by one and check if it gives a unique hit. (confirm chromosomal location of the gene from NCBI or other sources)
• If it gives multiple hits, select a different set of primers from Primer 3 Plus and repeat the same process until a unique set is obtained.

➢ Order primers through Invitrogen:

• Purification: Desalted
• Starting Synthesis Scale: 25nmole
• Ship Medium: Dry
• Normalization: None
• Calculate primers to 100uM using DNA suspension Buffer from Teknova cat # T0221

➢ Contact me if you have any questions (betul.kacar@biology.gatech.edu)

Primers for Kan

• Kan Cassette Test (Fwd): 5’ GTGTAGGCTGGAGCTGCTTCGAAGTTCCTATACTTTCTAG 3’
• Kan Cassette Test (Rev): 5’ ATTCCGGGGATCCGTCGACCTGCAGTTCGAAGTTCCTATT 3’

Site-Directed Mutagenesis

• pET15b map
• QuikChange II STM Kit Protocol
• QuikChange Primer Design (Agilent)

LTE Assays

araA Marker Test:

• Make sure to have: HaeII Restriction Enzyme (in the -20C Freezer RE Stocks box), NEB Buffer 4, and good luck
• araA marker revertant test

RT-PCR

• BioRad Applications Guide
• Green RNA to CT of AB
• mirVana mRNA Isolation Kit
• A helpful tutorial by Margaret Hunt
• Dunham lab has some very helpful tips on microarray & genotyping studies