1 reply to this topic

[nuramar]

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...

[Wunder]

Hello

I have minimal experience on animal tissue but I'll make some suggestions.

I'm wondering where you got this protocol from? Is this from the literature? Also did you design the primers yourself?

With a faint band the best thing to do is to cut the pcr fragment of the correct size, do a gel extraction and then clone it. This will dramatically amplify the number of copies of your pcr fragment. You can even send the plasmid for sequencing and this will generally give you a cleaner sequence than sending just the pcr fragment.

Hope that helps

Edited by Wunder, 08 April 2015 - 11:11 PM.