Hi Everyone,
I am trying to sub-clone TCF7 gene (transcript variant 1) into pCDH-CMV-MCS-EF1 Puro vector from some other commercially available vector by InFusion cloning(recombination). I got nice appropriate size specific band during PCR amplification, the positive colonies, I send it for sequencing and when blast I could see the match in DNA sequences. Interestingly, DNA sequences match from start to around 150 bps, then around 200 bps is missing and then rest all match up to Stop Codon. Could you anyone please suggest me why DNA sequences are missing from the inbetween the gene from the clone.
Thanks!
Abhishek
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Vector Database - pCDH-CMV-MCS-EF1-Puro
Did you start with gDNA or cDNA? If you started with cDNA and there is a 200 bp intron in your cloned region, then you would expect that region to be missing. You did say it's a "transcript variant" so I'm assuming you used cDNA.
The BLAST results default to comparing to the gDNA, which would show that your cloned sequence is "missing" a region compared to the gDNA.
Is this the correct sequence? https://www.ncbi.nlm.nih.gov/nuccore/NM_003202.5?from=227&to=1381&report=fasta
I'm seeing a lot of poly G and poly C regions in it, and I'm guessing that could be what the problem is, if the missing ~200 bp chunk is flanked by regions with a lot of C/Gs. Homologous recombination based cloning methods like InFusion and Gibson Assembly are prone to misassembling repetitive sequences.
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