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roshanbernard's Content

There have been 5 items by roshanbernard (Search limited from 23-July 18)


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#175291 Help in ACT(artemis) comparision tool

Posted by roshanbernard on 06 June 2015 - 12:24 AM in Bioinformatics and Biostatistics

i was bit early in posting this..:).. found a solution myself... if anyone needs this info... 

 

Go to this website 

 

http://www.webact.org/WebACT/generate

 

upload ur sequences... and in a while u can download the data... that contains all 3 sequences and 2 blast files(comparision files).. upload them in ACT and u can view them.. 




#175290 Help in ACT(artemis) comparision tool

Posted by roshanbernard on 05 June 2015 - 11:39 PM in Bioinformatics and Biostatistics

Hi,

i have three whole genome sequence from bacteria and i want to compare them. I am trying to Use ACT from artemis for the comparison. While i am trying to upload the files it is prompting me to use 1. comparison file and 2. sequence file for every bacterial WGS... Can anyone tell me whats comparision file and how do we generate it.

Thanks in advance

regards

rosh




#174440 Reducing contamination in 16s PCR for metagenomic library prer

Posted by roshanbernard on 16 April 2015 - 05:03 PM in PCR, RT-PCR and Real-Time PCR

yeah.. i think i will try reducing the PCR cycles and try it out. For the moment i use 34X... better i reduce and see the difference.. thanks again.. :)




#174437 Reducing contamination in 16s PCR for metagenomic library prer

Posted by roshanbernard on 16 April 2015 - 04:46 PM in PCR, RT-PCR and Real-Time PCR

Getting no-template controls for 16s PCR is probably almost impossible. Everything has some bacterial DNA, including, likely, you PCR reagents, water, tubes. But the amounts are very small, and are overwhelmed by the presence of a real template. If you can do qPCR, you will likely see that ampllfication happens only after many cycles. I would not worry about it. If you are concerned, sequence the no-template control band, and conclude that it is different from your template sample. To really pin it down without qPCR, do serial dilutions of your template and sequence the band from the dilution series.

Hi phage,

 

Thanks for your response. well, i did sequence the negative bands. And the blast hit varies from pseudomonas, e-coli and some unclultured etc. The main concern is i am dealing with Metagenomics using 16s Amplicons using Iontorrent. Hence if there is even little contamination, i believe that will be amplified. some one suggested me articzymes pcr de-contamination kit. However that works best with qPCR. anyays i am going to try with it.  i hope i can get rid of contaminations.

 

Thanks again for ur reply

 

regards

 

rosh




#174379 Reducing contamination in 16s PCR for metagenomic library prer

Posted by roshanbernard on 14 April 2015 - 05:27 PM in PCR, RT-PCR and Real-Time PCR

Hi everybody,

Recently i came across with the contamination with he PCR kits while i was trying to amplify the 16s Amplicons using V1-V2 primers. i have used Takara and Bioneer kits. But both of them gives a very prominent band even with the negative controls(primers only). i did the capillary sequencing for the negative bands, which showed the presence of some uncultured strains and e.coli.. etc....

I even tried treating them with Sau3A1. But then after de-activating Sau3A1, the taq polymerase becomes inactive....

Can anyone suggest me a good kit for the 16s Amplicon sequencing...

Please share ur experiences if anyone came across with such problems

Thank you

Rosh




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