Recipes & Protocols: Difference between revisions
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===Protocols=== | ===Protocols=== | ||
*[http://openwetware.org/wiki/Choosing_primers_for_qPCR Choosing primers for q-PCR] | *[http://openwetware.org/wiki/Choosing_primers_for_qPCR Choosing primers for q-PCR] | ||
===Primer designing for BUGS=== | |||
➢Get the gene name (eg: MYD88) | |||
*Go to NCBI (ncbi.nlm.nih.gov), and search in ‘Nucleotide’ for the gene sequence. | |||
*Select Homo sapiens 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. | |||
*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. | |||
➢Go to the UCSC genome browser website (http://genome.ucsc.edu/) and select the ‘BLAT’ tool. | |||
*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 Betül for questions (betul.kacar@biology.gatech.edu) | |||
Revision as of 09:23, 28 May 2013
Tools
Gene/Protein Info
Protocols
Primer designing for BUGS
➢Get the gene name (eg: MYD88)
- Go to NCBI (ncbi.nlm.nih.gov), and search in ‘Nucleotide’ for the gene sequence.
- Select Homo sapiens 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.
- 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.
➢Go to the UCSC genome browser website (http://genome.ucsc.edu/) and select the ‘BLAT’ tool.
- 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 Betül for questions (betul.kacar@biology.gatech.edu)
