Double-stranded sequencing of cDNA clones containing long poly(A) tails using anchored poly(dT) primers

ID: 83

Group:

Methods for DNA sequencing

Prepared by:

Bruce A. Roe (broe@uoknor.edu)

Detail:

Sequencing double stranded DNA templates has become a common and efficient procedure (10) for rapidly obtaining sequence data while avoiding preparation of single stranded DNA. Double stranded templates of cDNAs containing long poly(A) tracts are difficult to sequence with vector primers which anneal downstream of the poly(A) tail. Sequencing with these primers results in a long poly(T) ladder followed by a sequence which is difficult to read. In an attempt to solve this problem we synthesized three primers which contain (dT)17 and either (dA) or (dC) or (dG) at the 3′ end. We reasoned that the presence of these three bases at the 3′ end would ‘anchor’ the primers at the upstream end of the poly(A) tail and allow sequencing of the region immediately upstream of the poly(A) region.

Year prepared: NULL

Date uploaded: 2010-02-26 14:28:18

Laboratory Protocols

Select from:Additional methods & referenceAflatoxinsAssaying antifungal levelsAssaying mycotoxinsAztec Labs Diagnostic testing ProtocolsBiochemical CalculatorsBiofilmBiotypingCell Component ExtractionClinical BreakpointsCollection of Clinical MaterialCosmidsDetection in clinical samplesDNA extractionEnzyme synthesisFungal Molecular BiologyGeneral Molecular BiologyGeneticsGrowth and StorageHistologyMethods for DNA sequencingMicroscopyModel systems: in vitroModel systems: in vivoMore protocol collectionsPCR  -RT-qPCRPhagocytosisPlanar liquid chromatographyPost Antifungal Effects (PAFE)Preparation of amphotericin pasteProtein methodsProtocol overviewsQuality control in mycologySampling Aspergillus sporesSecondary metabolitesSpecies/Strain identification  -In situ hydridizationStandard Operating Procedures:National Aspergillosis CentreSusceptibility testingVeterinaryVideo Protocols at the FGSCWhole blood fractionationwhole-cell screening