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faint dna band after pcr purification


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#1 nuramar

nuramar

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Posted 15 December 2013 - 09:04 AM

Hi guys..

 

I really need someone to help me on this problem.I am currently working on bat tissue samples.The samples were taken from museum where they were preserved in 95% alcohol at room temperature for few years. We took the samples and  preserved in 95% ethanol stored in -20.We did genomic extraction using QIAGEN kit but fail to get any band.just smear...however, I try to proceed with pcr and still fail to get any band.After several methods of extraction, I finally manage to get pcr product(although this method showed smeared bands in genomic extraction) using CTAB+PCR purification method. Annealing temperature used was 63 deg. the size of the band is 700bp. I did purification using PCR purification kit but i got very faint band. Although i've tried using 35-40ul sample of PCR product,I still obtain faint band.The same thing happen when I use Gel extraction kit...I've tried loading 35-40ul pcr product in few wells.After purification, I try to run the sample again but the band is very faint. i sent the sample for dna sequencing, but fail. How am I suppose to send this sample for sequencing when the band is very faint? Please help me....thank you in advance...






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