JCAOligoTutorial1b-TmClarification: Difference between revisions
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=== How to analyze the 'secondary structure' of your oligos === | === How to analyze the 'secondary structure' of your oligos === | ||
When designing oligos, there is some ambiguity about whether you want a high or a low Tm, and exactly what is meant by Tm. First, let's disambiguate the term Tm, because there are | When designing oligos, there is some ambiguity about whether you want a high or a low Tm, and exactly what is meant by Tm. First, let's disambiguate the term Tm, because there are 3 equally important but different Tm values for the types of oligos you're designing in these tutorials. Let's consider the oligos in the first tutorial's example: | ||
'''Construction of KanR Basic Part Bca9128''' | |||
<pre> | |||
PCR ca1067F/ca1067R on pSB1AK3-b0015 (1054bp, pcrpdt) | |||
Digest pcrpdt (EcoRI/SpeI, 1038+11+5, L, pcrdig) | |||
Digest pSB1A2-I13521 (EcoRI/SpeI, 2062+946, L, vectdig) | |||
Ligate pcrdig and vectdig (pSB1A2-Bca9128) | |||
----------------------------------------- | |||
>ca1067F Forward Biobricking of KanR of pSB1AK3 | |||
ccagtGAATTCgtccTCTAGAgagctgatccttcaactc | |||
>ca1067R Reverse Biobricking of KanR of pSB1AK3 | |||
gcagtACTAGTtccgtcaagtcagcgtaatg | |||
</pre> | |||
Specifically, let's focus on oligo ca1067F. Let's modify that oligo to introduce a point mutation into the annealing region of this oligo. I'll put the mutated base in bold so you see what I did. This mutation ultimately is silent: | |||
ca1067F ccagtGAATTCgtccTCTAGAgagctgatc'''G'''ttcaactc | |||
Revision as of 17:48, 24 January 2012
How to analyze the 'secondary structure' of your oligos
When designing oligos, there is some ambiguity about whether you want a high or a low Tm, and exactly what is meant by Tm. First, let's disambiguate the term Tm, because there are 3 equally important but different Tm values for the types of oligos you're designing in these tutorials. Let's consider the oligos in the first tutorial's example:
Construction of KanR Basic Part Bca9128
PCR ca1067F/ca1067R on pSB1AK3-b0015 (1054bp, pcrpdt) Digest pcrpdt (EcoRI/SpeI, 1038+11+5, L, pcrdig) Digest pSB1A2-I13521 (EcoRI/SpeI, 2062+946, L, vectdig) Ligate pcrdig and vectdig (pSB1A2-Bca9128) ----------------------------------------- >ca1067F Forward Biobricking of KanR of pSB1AK3 ccagtGAATTCgtccTCTAGAgagctgatccttcaactc >ca1067R Reverse Biobricking of KanR of pSB1AK3 gcagtACTAGTtccgtcaagtcagcgtaatg
Specifically, let's focus on oligo ca1067F. Let's modify that oligo to introduce a point mutation into the annealing region of this oligo. I'll put the mutated base in bold so you see what I did. This mutation ultimately is silent:
ca1067F ccagtGAATTCgtccTCTAGAgagctgatcGttcaactc
