Search found 81 matches

by roniadam
Sat May 15, 2010 9:39 am
Forum: Molecular Biology
Topic: Western Blotting of small molecular weight
Replies: 4
Views: 9415

Western Blotting of small molecular weight

Hi Guys I have done WB for my proteins (~ 5 and 17 kDa) using biorad's gradient precast Tris/Glycin gels and was not able to detect my proteins. I would like to know what voltage should I have for unning the gel and what voltage and duration should I use when transfer to PVDF membrane using Biorad's...
by roniadam
Sat Apr 24, 2010 2:35 am
Forum: Molecular Biology
Topic: micromolar conversion
Replies: 3
Views: 13486

Hi
Thank you very much for your kine help
by roniadam
Thu Apr 22, 2010 6:19 am
Forum: Molecular Biology
Topic: micromolar conversion
Replies: 3
Views: 13486

micromolar conversion

Hi All

I am getting stuck

My primer concentration is 0.1 uM in 10 uL and I need to get 125 ng of this primer.
how many uL should i take from my primer in total reaction of 50 uL.

Is there any online calculator that can help me ???
by roniadam
Wed Apr 14, 2010 3:06 pm
Forum: Molecular Biology
Topic: sample preparation for SDS-PAGE
Replies: 2
Views: 2797

sample preparation for SDS-PAGE

Hi

I got confusion about the function of Laemmli sample buffer....
Does Laemmli sample buffer lyse adherent cells for SDS-PAGE? or I need to lyse my cells first with a cell lysis solution and then dilute the cell lysate with laemmli buffer?
by roniadam
Tue Mar 16, 2010 12:24 pm
Forum: Molecular Biology
Topic: Western Blotting protocol using BioRad
Replies: 2
Views: 2185

No step by step instruction found
by roniadam
Mon Mar 15, 2010 1:23 pm
Forum: Molecular Biology
Topic: Western Blotting protocol using BioRad
Replies: 2
Views: 2185

Western Blotting protocol using BioRad

Hi every one

I am new to protein analysis field and want some help. I bought all reagents from BioRad required for western blotting. I need a step by step protocol (in detail) please to start runnig this assay. Can any one help me please using the biorad reagents and equipment???
by roniadam
Fri Nov 27, 2009 11:12 pm
Forum: Molecular Biology
Topic: mRNA stability assay by actinomycin
Replies: 0
Views: 4349

mRNA stability assay by actinomycin

Hi I want to study the stability of mRNA by adding Actinomycin D on transfected cell line. What is the suitable time to treat the transfected cells with Actinomycin to inhibit RNA synthesis? Is it after 24 hrs posttransfection or more? I did a first trial at 16 hrs posttransfection using 5 microgram...
by roniadam
Wed Nov 25, 2009 3:10 am
Forum: Molecular Biology
Topic: role of donor vector in gene cloning
Replies: 2
Views: 2014

Thanx Jack

you r right. I am talking about the gateway system. Thank u for your very useful info.
by roniadam
Sun Nov 22, 2009 10:30 am
Forum: Molecular Biology
Topic: role of donor vector in gene cloning
Replies: 2
Views: 2014

role of donor vector in gene cloning

Hi

I would like to know why do we use a donor vector when we clone a desired gene to introduce it into a destination vector for expression analysis? why we don't go directly to introduce our gene into the destination vector without recombining it into the donor vector?
by roniadam
Sat Oct 10, 2009 9:15 am
Forum: Molecular Biology
Topic: nested pcr
Replies: 3
Views: 2327

I had the same problem before. I advice you to use an adjuvant to improve your PCR effeciency. I used Q solution that comes with hotstar PCR master mix and it gives me a very good amplicon.
by roniadam
Wed Oct 07, 2009 5:28 am
Forum: Molecular Biology
Topic: Electroporation of human bladder carcinoma 5637 cell line
Replies: 3
Views: 2191

Hi JackBean:

I was not able to find any paper explaining the electroporation protocol for 5637 cells. all papers I saw are transfecting the 5637 using Lipofectin or Fugene. but no paper found explaining the electroporation ptotocol.
by roniadam
Tue Oct 06, 2009 5:25 am
Forum: Molecular Biology
Topic: Electroporation of human bladder carcinoma 5637 cell line
Replies: 3
Views: 2191

Electroporation of human bladder carcinoma 5637 cell line

Hi Guys,

I am searching for the protocol of human bladder carcinoma 5637 cell line using electroporation technique. I mean what voltage, pulses and cuvette should I use for susccessful transfection? I use Genepulser machine from BioRad.
Any help please?