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False positive in empty wells in RNA dot blots

RNA Dot blot

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#1 marek82313

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Posted 26 March 2015 - 11:05 AM

Dear all,

 

I had a weird results with a dot blot prepared with RNA samples.

Briefly:

- We prepared two dot blots in parallel with the same RNA samples,

- each sample was dotted twice on the same filter, one time the RNA was treated with RNase A as negative control, one time the sample was mocked treated,

- the two filters were hybridized with two different radioactive probes, one filter was hybridized with the probe for the target gene, the second filter with the probe for the control gene. Results were consistent,

- after some months (8 months) the filters were checked for radioactivity, they were cold, no signals were detected,

- the filters were hybridized again but the probes were swapped (i.e. the first filter was now hybridized with the probe for the control gene and viceversa),

- the filter now hybridized with the probe for the control gene (previously was the target gene) gave consistent results. The filter that was previously hybridized with the probe for the control gene and now was hybridized with the probe for the target gene was weird: the first wells of the blot (the uppest ones) had a very faint signal, those at the bottom of the filter had a very strong signal, the problem is that the signal is detected also in wells were no sample was loaded. I can exclude to have exchanged the filters because none of the filters we prepared contained samples at the same position of the supposed empty wells.

 

Have someone ever experienced a weirdness like this?

 

Please find attached a picture of an autoradiographic film, the empty wells are supposed to be the three at the bottom on the right.

 

DSC_1205.jpg



#2 purplehayes

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Posted 11 April 2015 - 07:43 AM

Hi, 

 

I am new to this forum and I have a question. My question regards the actual suctioning of samples onto the membrane using a dot blot apparatus. Have you had any difficulty getting the samples to properly suction from the wells onto the membrane? Everything will suction just fine other than the samples themselves (fairly highly diluted RNA). I have gotten this to work with inconsistent results correlating with variable rate of suction speeds. I have been hydrating the membrane prior to dot blot apparatus assembly, full suction during assembly, have used filter paper underneath the membrane, have not used filter paper under the membrane, have used buffer (DEPC water) prior to sample application for membrane rehydration, have used buffer for unused wells to increase suction in the sample wells, have elevated the entire apparatus above vacuum level, and the troubleshooting list goes on. I have been getting diffuse signals indicating improper suctioning. If you have some advice I am all ears! Please let me know!

 

thank you!



#3 marek82313

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Posted 11 April 2015 - 08:08 AM

Hi purplehayes,

 

sometimes some of the samples get sucked more slowly than other, usually we just wait a little more. In case the sample seems "stuck", I usually take a Gilson pipette (P1000) and I pipette the sample in the well that has some troubles. Usually, after a pipetting the sample get sucked as usual. Might it be a problem related to the "strength" of your vacuum apparatus? 

 

Bye







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