Sam:LabNotes/Microbione/2009-2-3: Difference between revisions
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>Sam Chiang No edit summary |
>Sam Chiang No edit summary |
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#Collect the human S18 cDNA sequence | #Collect the human S18 cDNA sequence | ||
#Clean the sequcne and mask the inconsistat nucleotide | #Clean the sequcne and mask the inconsistat nucleotide | ||
#Paste the sequence on KZ's Primer3 calculator[http:// | #Paste the sequence on KZ's Primer3 calculator[http://arep.med.harvard.edu/kzhang/cgi-bin/primer3_www.cgi] | ||
#Criteria: | #Criteria: | ||
Primer size: Min: 18bp, opt:20 bp, Max: 22bp | Primer size: Min: 18bp, opt:20 bp, Max: 22bp | ||
Product size: ~200 bp; ~300 bp | Product size: ~200 bp; ~300 bp | ||
Annealing Temp: Min:57, Opt:58, Max:59 | Annealing Temp: Min:57, Opt:58, Max:59 | ||
#Using Netprimer[http://www.premierbiosoft.com/netprimer/index.html]to evaluate primer structure. | |||
#Pick up two primers with differnt size of amplicon. | #Pick up two primers with differnt size of amplicon. | ||
>h18S_211_f | >h18S_211_f | ||
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>h18S_211_r | >h18S_211_r | ||
CATTATTCCTAGCTGCGGTA | CATTATTCCTAGCTGCGGTA | ||
----------------------------------------------- | |||
>h18S_306_f | |||
GTACAGTGAAACTGCGAATG | |||
>h18S_306_r | |||
CGACTACCATCGAAAGTTGA | |||
----------------------------------------------- | |||
Revision as of 06:38, 9 February 2009
Primer design for human 18S and Bacteria 16S
Objective
Besides the realtime monitoring,
- Design 18S primers for positive control / validation purpose of a successful MDA reaction using human genome template.
- Design 16S primers for positive control / validation purpose of a successful MDA reaction using bacteria genome template.
- These primers could also be used to detect the contamination of exogenus gDNA.
Human S18 primer
- Collect the human S18 cDNA sequence
- Clean the sequcne and mask the inconsistat nucleotide
- Paste the sequence on KZ's Primer3 calculator[1]
- Criteria:
Primer size: Min: 18bp, opt:20 bp, Max: 22bp Product size: ~200 bp; ~300 bp Annealing Temp: Min:57, Opt:58, Max:59
- Using Netprimer[2]to evaluate primer structure.
- Pick up two primers with differnt size of amplicon.
>h18S_211_f TTGCTGCAGTTAAAAAGCTC >h18S_211_r CATTATTCCTAGCTGCGGTA ----------------------------------------------- >h18S_306_f GTACAGTGAAACTGCGAATG >h18S_306_r CGACTACCATCGAAAGTTGA -----------------------------------------------