Discussion

4 months ago

Keith L. Morrison Canada

2

Votes

2

Comments

siRNA/RNAi/miRNA transfection human

I would like to regulate the expression of a gene and in order to do that, I have purchased specific siRNA. After optimizing my transfection protocol and using electroporation I have achieved a 60-70% reduction of the gene of interest. However, I cannot observe a significant reduction of mRNA expression but only a reduction of protein. What might be the problem? Could the problem be in my cell treatment method?

Experiment: siRNA / RNAi /miRNA transfection Human

Discussion

4 months ago

Stefan Fuhrmann Germany

1

Vote

2

Comments

Will presence of EDTA effect cDNA synthesis

I intend to use iScript cDNA Synthesis Kit in order to synthesize cDNA for qPCR. I have confirmed that my RNA is pure however, according to my extraction protocol I have suspended the RNA in TE buffer containing 1mM EDTA. Will the presence of EDTA have an effect on cDNA synthesis?

Experiment: cDNA synthesis Bacteria

Discussion

3 months ago

Paul G. Macon United States

3

Votes

1

Comment

RNA isolation from tissue

How do I extract RNA from animal tissue without using liquid nitrogen? I tried the RNA extraction by using the TRIzol reagent and I homogenize the tissue using polytron homogenizer at room temperature for 30secs is this correct?

Experiment: RNA isolation Tissue

Discussion

3 months ago

Mario Udinese Italy

2

Votes

1

Comment

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Experiment: CRISPR Mouse

Discussion

3 months ago

Milena Alexeyeva Russian Federation

2

Votes

1

Comment

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

Experiment: CRISPR Human

Discussion

4 months ago

A.C.Burton United Kingdom

1

Vote

1

Comment

Multiple gene silencing using ShRNA

Hello, can someone here help me? I am trying to silence e-selectin and ICAM-1 in endothelial cells. I would like to know if this is possible using shRNA

Experiment: shRNA gene silencing Mouse

Discussion

4 months ago

M. Daecher Germany

1

Vote

1

Comment

Live/dead assay Bacteria

Hello everyone! I am going to do a live/dead assay for my cells and I saw that I can use both fluorescence and absorbance as my detection method. Is there a difference in the results depending on the method? Is one method preferred over the other in certain situations?

Experiment: Live / Dead assay bacteria

Discussion

4 months ago

R. Verma India

1

Vote

1

Comment

DNA isolation column clogged

During centrifugation, the column got clogged and I was unable to continue with the protocol. How can I unclog it?

Experiment: DNA isolation / purification Cells

Discussion

8 months ago

Maryam Amir Qatar

2

Votes

1

Comment

When should I add the polymerase for hot start PCR?

I keep getting a non-specific band in PCR so I would like to try a hot start PCR manually. How should I prepare the mix and at which step should I be adding the polymerase?

Experiment: PCR Hot start PCR

Discussion

8 months ago

Vitoria Rocha Almeida Brazil

1

Vote

1

Comment

Will I be able to ligate blunt ends without the presence of ATP?

Will I be able to ligate blunt ends without the presence of ATP?

Experiment: DNA ligation

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