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seanspotatobusiness's Content

There have been 5 items by seanspotatobusiness (Search limited from 22-September 19)

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#179204 How clean do I need my DNA to be?

Posted by seanspotatobusiness on 18 May 2017 - 05:14 AM in PCR, RT-PCR and Real-Time PCR

I have been using kits with columns to purify genomic DNA which I then use as a template for PCR for sequencing or T7E1/CelI mutation-detection assays. I accidentally ordered a kit which does not use columns but rather the older approach of chloroform and ethanol precipitation. I think it should be fine but then I wonder why I've been using spin columns... just for convenience? 

#179147 Where to gate with overlapping population?

Posted by seanspotatobusiness on 14 April 2017 - 06:28 AM in Flow Cytometry

My fluorescent cells are only weakly fluorescent so they overlap with the non-fluorescent cells as in the below image. Would it be acceptable to move the gate to the left to get more of the fluorescent cells and then simply subtract 2% (or whatever value shows positive in the negative control) from all the values to obtain a good indication of the percentage of "stained" (they're not stained, they're expressing a fluorescent protein) cells?



#179123 How long do you reckon a cell pellet can survive before it must be resuspended?

Posted by seanspotatobusiness on 04 April 2017 - 04:16 AM in Tissue and Cell Culture

How long do you reckon a cell pellet can survive before it must be resuspended? I figure it limits how large a batch of samples can be processed at the same time.

#179033 Three experiments with three replicates in each; how to apply calculate SEM?

Posted by seanspotatobusiness on 21 February 2017 - 06:19 AM in Bioinformatics and Biostatistics

I have done the same experiment three times and in each experiment the same thing was done three times; thus I have nine data points for every sample. Should I average the three within-experiment-replicates and use those three values find a super-average and calculate an SEM or should I use all nine values to calculate an average and SEM? Obviously the average will come out the same but the SEM won't.


I think I should use the SEM calculated from the three averages because there were three experiments and the three replicates within each experiment were not really separate because they were being done together. This also reduces the value of the SEM which I think makes the data look more reliable.


I made up some data to produce an example.



#178939 Increasing lipofection efficiency by agitation?

Posted by seanspotatobusiness on 16 January 2017 - 08:10 AM in Tissue and Cell Culture

Has anyone else tried this? For me, agitation did not improve efficiency for Lipofectamine 3000 or DMRIE-C. There's a magazine article about it here but I couldn't find much else about it online with my lazy, half-assed search.

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