My sequence doesn’t match Addgene’s sequencing result, what should I do?Ĭheck your trace file first the apparent mismatch/mutation may be the result of a mis-called peak in the trace file. The corresponding primer sequence used for each reaction.The original trace file (.ab1 file) provided by your sequencing company or core facility.If you obtain a different sequence than expected and wish to contact Addgene about the accuracy of your plasmid, please email and include: This is an example of a trace file from a high-quality portion of a sequencing reaction: You should receive your sequencing results as a trace file (.ab1) which graphically depicts the sequence as a series of colored peaks corresponding to one of the four nucleotide bases. If you require additional sequencing and need to design a custom primer, Addgene recommends using Addgene's sequencing results as a reference for primer design.Ī good sequencing reaction will produce between 300-900 base pairs of useable sequence. Is your custom primer found within the Addgene sequencing results? What primers were used by Addgene during quality control?Īddgene lists the primers used to obtain each result above the posted sequence in the "View Sequence" link. Many sequencing cores have a list of common primers that can be requested for no additional charge. There are many widely used, common primers that are often found within the plasmid backbone (See Addgene's Sequencing Primers for reference). The primer should be a minimum of 50 nucleotides and a maximum of 300 nucleotides from your target.Īre there commercially available primers that can be used for sequencing? Is the primer an appropriate distance from the target? Make sure the primer only anneals once within the entire construct. cotr araBAD promoter, forward primerįor vectors with E.There are a number of factors to consider when selecting sequencing primers, including: cerevisiae GAL10 promoter, forward primerģ' end of Gal4 DNA binding domain, forward primerģ' end of Gal4 activation domain, forward primerįor vectors with E. Human elongation factor-1a promoter, forward primer Human CMV immediate early promoter, forward primerĥ' end of Cre recombinase, reverse primer The 1st readable base starts about 50 bases downstream of the primer binding site.Īvailable Common Primers (Complete List in Excel) Nameįor Pichia vectors with AOX1 terminator, reverse primerįor Pichia vectors with AOX1 promoter, forward primerĪlpha factor signal sequence, forward primerĥ' end of ampiciltrn resistance gene, reverse primerĥ' end of chloramphenicol resistance gene, reverse primer.Avoid multiple binding sites on the template: putative binding sites are defined as 80% of your primer sequence or 100% of last 7 bases on the 3' end aligned to another site on the template.Avoid secondary structures: hairpins, palindromic sequence, dimers, and more 3Gs or 3Cs in a stretch.What tips do you have for designing primers? The agar should not be too liquid for shipment. Please use endA- strains such as Dh4α, HB101, and XL1-Blue. E.coli strains can be shipped as single colonies on agar plates.You can find detailed information on the Sample Preparation page. These plasmid can be concentrated by ETOH precipitation, centricon spin column, or use our template amplification service (RCA). Low copy number plasmids need to be further concentrated before sequencing.Please consult your special supplier for further information. Plasmid DNAs purified through most commercial kits are suitable for DNA sequencing.How do I prepare plasmids for sequencing? We strongly recommend to resuspend template or primer in 10 mM Tris (pH 8.5) or water. Should I resuspend my samples in water?Īvoid EDTA in submitted template or primer solutions. What tips do you have for designing primers? How should I submit samples? TypeĠ.5ug plasmid DNA + 3pmol primer in 12ul dH2Oġ00ng/1000bp purified PCR product + 1pmol primer in 12ul dH2Oġ00ng/1000bp unpurified PCR product in 10ul dH2O Oligo Calculator, calculate Tm, GC content, and Molecular Weight of your oligoĪ primer database for human and mouse genes.
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