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I am in the process of making RNA probes (900bp) for in situ. I have cloned my gene (DUSP 5) which 900 bp into a pBsk vector. I have sequenced the gene using M13 F/R, T3/ T7 and my own designed primers. When I sequence from the '3 end I get perfect match to my gene. However, when I sequence from the '5 end, using any of the three above primers, I don't get a match. I get stretches of 4-7 matching bp peppered between non-matching sequences. When I sequence from the '5 end all three primers give me the same sequence, so it's not random Ns and its not the primers. I also looked at the chromatogram of the sequencing results and it looks real. Does anyone have an idea how this is possible? Any help appreciated. Thanks.
You cloned the gene from cDNA (i.e. mRNA) or from genomic DNA? If the latter, then you may be looking at a mix of intron/exon sequence. There may be variant mRNAs expressed and you may have cloned a variant slightly different than the one you were looking for. Or you may have cloned a related gene by mistake—although, if the N-terminal sequences are identical for hundreds of bp, that is unlikely to be the case.
Must have been some funny business going on during the pcr, then. At least that's all I can think of. I would try either a different polymerase or maybe modifying the pcr protocol--higher temps, with or without Mg, etc. I like hot-start, high-fidelity enzymes like KOD or pfU polymerase. Sounds like you should just make another cloning attempt.
First, I am not sure why you subcloned this sequence into your final vector if you were aware of the problem after sequencing TOPO 10. The only explanation that I can come up with is that you accidently got a rare double ligation: your insert and random amplicon of the same size into your vector. However, it seems to me that you should have found out about that from your sequences since with 900 bp insert you should have vector sequence present on both sides. Additional data would be helpful.
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