Biochemical Techniques

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When running an SDA PAGE of a powdered form of recombinant protein, in what solution do I need to dissolve it in before mixing it with loading buffer?

I have a recombinant protein interleukin as a powder that I need to run in an SDS PAGE of a required concentration. Before mixing it with loading buffer how should I liquefy to get the required...

10 October 2014 5,447 4 View

What is the best detection system for western blots < $10K?

I am preparing a grant proposal and need to find a detection system for western blots for less than $10K. I really want a Li-Core Odyssey CLx but can't fit it in the budget. The Li-Core Fc is 'low...

10 October 2014 450 3 View

How can I isolate double stranded DNA from single stranded in a mixture?

Please suggest any protocol which is cost effective and feasible. Thanks in Advance.

10 October 2014 6,916 5 View

Why does one PCR band after gene clean become four bands?

Hi, I run my PCR product on agarose gel, cut my 3kb target band and gene clean it. But when I run my gene clean product on agarose gel, apart from my 3kb target band, it also gives me another...

10 October 2014 8,223 7 View

Could anyone tell me the best parameters for transfer of a 7kDa protein? Nitrocellulose vs PVDFat 2uM?

What is the optimum voltage and ampres? Thank you all in advance.

10 October 2014 739 3 View

Does anyone know how miR-21a induces the expression of total proteins?

Now I start my exp with miR-21a and pdcd4. Although many researchers have studied the relation about miR-21a and PDCD4,there is no paper pointing to the changes of total proteins when me...

10 October 2014 5,506 4 View

How can I avoid antibody chain bands on western blot?

I want to detect phosphorylation signals (pAkt, p-p38, pERK, pJNK, p65, etc) in T cells after being stimulated with hamster anti-CD3 and hamster anti-CD28, then began to calculate time after...

10 October 2014 6,928 8 View

Sequence the coding regions of a 14 exons - gene

Dear all. I'm trying to sequence the coding regions of a gene called NDST1 in a 400 individuals - population ( The samples of the population were collected in total DNA form). Problem is the gene...

10 October 2014 6,935 11 View

Does cell sorting by Flow Cytometry trigger primed macrophages?

We noticed that macrophages from anti-CD40 injected mice, after we sort them, became very activated so that they don’t need another stimuli (TLR4 or TLR9 agonists) to produce high levels of nitric...

10 October 2014 1,300 3 View

How can I proceed to carry out an MS-PCR to genotype a polymorphic gene in Plasmodium falciparum?

I'd like to genotype a gene in plasmodium falciparum gene, which method can use that doesn't require RFLP. Which MS-PCR can I use?

10 October 2014 9,118 1 View

Am I getting false positve qPCR reactions due to proximity of positive controls?

We are getting numerous false positive qPCR reactions in wells adjacent to our positive controls. We are using a Bio-Rad CFX Touch 96 with Bio-Rad plates and cover seals. At first it was...

10 October 2014 4,555 5 View

Is anyone familiar with PENTR / D -TOPO cloning?

I have been trying to generate clones using pENTR DTOPO for gateway cloning. I am able to amplify my insert, set up a reaction and get colonies. But when I do a colony PCR with insert specific...

10 October 2014 9,541 8 View

Is anyone familiar with cloning vectors?

I am cloning a gene into a vector that has the reporter gene LacZ, but I am getting some background and almost no clones. If I use another enzyme, to cut the reporter gene LacZ, would it be a good...

10 October 2014 10,180 1 View

Why am I getting an abnormal qPCR amplification plot?

Hello everyone! I am using AB Stepone system with Qiagen QuantiNova PCR kit. After doing my first trial, my qPCR results were very strange, the plot are "hook"-shaped (attachment). Anyone know...

10 October 2014 7,372 7 View

Does any body know the difference between ficoll and percoll? Can I use percoll for isolation of the PBMC from whole blood and to use it?

Any body know the difference between ficoll and percoll? Can I use percoll for isolation of the PBMC from whole blood and to use it?

10 October 2014 1,231 6 View

What is the percentage (%) amount of 18β-glycyrrhizic acid in Total Glycyrrhic acid?

I have a procedure for analysis of total Glycyrrhic acid in Liquorice Dry Extract. But I want to evaluate 18β-glycyrrhizic acid. Can anybody tell me what is the percentage content of...

10 October 2014 1,944 2 View

Is it possible to remake cDNA from RNAs left in the cDNA solution?

By mistake I took concentrated RNA (5ug) for cDNA synthesis(20ul reaction mix) and cDNA synthesis didn't happen. When I ran 2ul of that cDNA (after diluting to 50ul) in a gel, I could see RNA...

10 October 2014 1,469 6 View

How can I synthesize my protein without any extra residues?

I am using computational methods to design proteins that fold into specific shape. However, I found that, in reality, proteins after synthesis usually have tags (eg His tag) and liners, which...

10 October 2014 8,635 8 View

How much total protein can you get from exosomes which isolate from 1ML serum by UC?

Is there anyone who knows how many ug of exosomous protein I can get from 1ML serum. I isolated the exosomes by the methods of UC 3 times,and collected protein from 500ug,100ug and 400ug of 1ML...

10 October 2014 2,691 1 View

Is anyone familiar with BrdU / BrUTP incorporation?

I have a query on BrdU/ BrUTP incorporation in culture cells. Why is transfection/microenjection required for BrUTP incorporation. Whereas, We add BrdU directly into the culture medium to pulse...

10 October 2014 5,727 7 View

What is the process of calculating Rf factor from SDS-PAGE after protein extraction?

protein extraction

10 October 2014 2,877 2 View

How do I initiate polyacrylamide gel polymerization while using low bis-acrylamide concentration?

I am trying to create a polyacrylamide gel at low bis-acrylamide concentration (

10 October 2014 5,358 9 View

Is anyone familiar with pcr primers storage?

Hi everybody : Please : Can anybody tell me how long can I store the PCR primer solution under 20C ? Are there any additives or methods which can improve the primer stability during the storage ?

10 October 2014 5,839 16 View

Has someone experienced false negative results in colony PCR?

Hi all. I picked 12 colonies from agar plate and performed colony PCR. It showed no bands on the agarose gel. So I isolated and purified the plasmid and then digested it with the same restriction...

10 October 2014 3,318 4 View

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