Ravi Shankar Gautam

I am trying to visualize a ChIP seq data using the online UseGalaxy tool. I have proceeded with all the necessary steps required for Pileup Aligned files prior to Visualize, even Pileup has been generated for my Bowtie aligned file. But unfortunately, whenever I am trying to do Pileup Interval this tool is showing Error (Syntax Index Error) although I have sent back the error report to the team.

Can anybody help me out to proceed with the Pileup generate step?

Hello Every1!!!

I am trying to purify my MBP fused protein by affinity chromatography using amylose resins but getting to many non specific bands.. therefore i tried gel filtration but again and again i am getting few non specific bands, as i have to for crystal and assays i need to get a single band but after so many attempts I am getting non specific bands..

any suggestions for removing those non-specific bands.

My mbp fused protein is of 96.5 kda and I m getting non specific bands near about 62 kda...

I have attached 1 of my sds-page image of fractions eluted after GPC...

Actually i want to know is it acceptable to get variation in expression of housekeeping genes like GAPDH and Actin which i am getting in Real time pcr? in normal condition actin's avg ct is around 16 and that of GAPDH it is around 20,, but avg CT of GAPDH gets change whenever i m trying to do Real time at different condition, its aroung 16 whereas for actin it is nearby 14..

It this change in Ct of control (Housekeeping genes) are acceptable?

Actually I m concern about the maximum and minimum amount of cDNA we can use for Real time PCR, as we Rt pcr is very sensitive and can detect a very low amount of cDNA also.

Generally it is recommended in cDNA synthesis kit that we should use around 1-2ug of RNA for cDNA synthesis, bt with this amount i m not getting any amplication for my few specific genes except my controls i.e Actin and GAPDH and few other genes. Any reason for this???

Hi all,

I m very much confused for a double digestion reaction to check a fallout of 166 bp which has been cloned in 6900bp vector.

I have took around 1500 ng of my recombinant plasmid(6900+166 bp) as a template for a 20ul digestion reaction but after loading the complete reaction volume I didnt able to see any fallout in a 1% agarose gel . The vector backbone is quite bright and can be seen in a single band but thr is no band of 166 bp.

I have previously done colony PCR for my clone confirmation and it has shown a clear band of 166 bp.

any suggestion regarding this problem?

In order to do a cloning I am trying to do double digestion of my vector with Sph1 and Not1 enzymes , the sites are 14

bases apart. My question is - is it appropriate to do double digestion at a time with fast digest enzymes (Thermo) which mainly have a common universal buffer or should I go for sequential digestion with the enzymes? my concern is - as both the sites are nearby will be there any interference during the double digestion using both enzyme at a time?

Thank you

Hello all,

I am looking for bioinformatic tool/websites to know the location of fungal genes, as most of the genes are uncharacterized , is it possible to get their location on chromosome?

My organism is Rhizopus delemar/oryzae.

Hello all!

I am doing SDS-PAGE for my protein of predicted size of 53.5kda bt unexpectedly I am getting bands of around 60kda.. I have done this 3-4 times and Everytime the required band is about 60kda in induced samples.. I have sequenced my cDNA clone also and it is showing right sequence.

suggestion will be highly appreciated..

Thanks

I want to ask "does elution buffer provided with kits like gel elution/pcr purification etc interfere with any cloning processes like restriction digestion and ligation? as they contain salt and so as restriction digestion buffer and ligation buffer..