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what may have gone wrong?


21 replies to this topic

#1 yobou

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Posted 05 June 2018 - 06:42 PM

dear all

please check the attached image and let me know what may have gone wrong during PCR? bands are strangely diffuse 

thanks

Attached Files



#2 OldCloner

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Posted 23 July 2018 - 10:27 AM

Looks like you are getting non-specific amplification and possibly primer-dimers as well. You really need to tell us the size of the band you expect and the exact ladder you are using, or at least the size of the smallest band in the ladder.  Unless you give us more clues to go on, like what polymerase (kit) you are using, the lengths and Tms of your primers, and whether they are degenerate or nested, novel or something that has been used before, and what your template DNA is, we won't be able to help you much. We'd really like to help, but if you don't provide enough information, most people will just skip your question.



#3 yobou

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Posted 23 July 2018 - 12:13 PM

kit used: MyTaq™ Red Mix - Bioline

ladder: Nippon genetics 100 bp ladder, smallest band =100 bp

amplicon size: from the left: first 4 lanes ~150 bp; remaining lanes ~270 bp

primers used: previously reported 

reaction mix total volume: 20 microL

loaded amont on the gel: 20 microL

template source: RNA from in vivo mice samples



#4 OldCloner

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Posted 26 July 2018 - 05:42 AM

Thanks for the answers. So your RNA template was converted to cDNA by reverse transcriptase (RT), I assume. Was it total RNA or did you purify mRNA?  Do you expect the target to be highly expressed or sort of weakly expressed? If it is a rare target, that increases the chance that primers will go astray and amplify some false but common target if amp conditions are not quite right.

 

I will mention this, although I don't think this is your problem: There is some possibility that there is contaminating genomic DNA in your RNA preps, though many RT kits have a DNAse step to reduce this possibility.  Good primer design usually places primers so that they cross  an exon-exon splice site; if they stick to genomic DNA contaminants the product (with intron) would be too large to amplify under the conditions used. If you are using well-characterized primers that should have been taken care of.

 

Have you seen a published gel figure from the researchers who designed the primers?  Did it look better, with one single band per target? You probably need to try adjusting your amp conditions (template amount, annealing temp, extension time, etc.) to try to eliminate the extra banding and what appears to be a fair amount of either primer-dimer or leftover unincorporated primers (the fuzzy part near the bottom of each lane). If you are following a published protocol but have to do something different (like use a different thermocycler) you often have to make adjustments.  Hint- adjust does not always mean "add more."  Sometimes putting in less primer or template actually helps.  In my experience, if I amp 20 ul rxns I only need to run 5 ul on a gel, so you may be overloading the gel a bit, too. All the salts n' stuff in the PCR rxn can cause distortion in the gel run itself., and yours is a bit "smiley." I know your kit already has loading dye in it. I've used something similar under a different brand name, and I usually do it for presence/absence assays. I do 5 ul rxns and load the whole thing. 

 

You can also try sequencing the PCR products and see if you get mixed sequence.

 

Hope something here helps.


Edited by OldCloner, 26 July 2018 - 05:44 AM.


#5 merlav

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Posted 30 July 2018 - 11:06 AM

I also would add that the gel have some electrical issues.  What voltage was used?? How many runs have being done with that buffer?  If re-use too many times it won't conduct properly the electricity.  I concur that loading 20µL on a gel is too much, better between 5-8µL unless is a very rare event or that the sample will be extracted from gel.  Did you ran a quality control of the samples?? If no, then next time I would suggest that you run all samples with 1 housekeeping gene.  Other thing to take in consideration is Mg concentration, too much concentration will make extra bands to appear.  


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I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
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#6 yobou

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Posted 30 July 2018 - 11:28 AM

I usually use freshly prepared running buffers each time and at a low voltage (50) . If I used 10uL loading volume the signal is weak. The wells of the gel are large enough (because of the comb!) and can accommodate upto 25uL.

today I got new chemicals (new boric, new EDTA). My previous EDTA was tetrasodium salt, but the new one is disodium salt and I am stuck with dissolving it. warming and stirring was insufficient to force it into solution. I heard about people autoclaving it but I am worried about degradation. can you let me know your method for dissolving disodium EDTA salt?  



#7 yobou

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Posted 31 July 2018 - 12:52 AM

finally EDITA disodium salt dissolved by pH adjustment 



#8 merlav

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Posted 31 July 2018 - 04:49 AM

Yes, EDTA only dissolves when it reaches the right pH, no need to heat. Running buffers don't need to be autoclave. Probably the specks and weird bands of the ladder that I'm seeing is un dilute reagent that I mistook for old buffer and or too high voltage. Yes the well can accommodate a lot of product, but the smear tells me that the product loaded is too much.   May I ask why you are doing an end point PCR instead a QPCR? If product is a so rare event a semi-quantitative PCR won't give the best results.  I do end point PCRs on cDNA samples just for quality control where I run samples RT+ and RT-(no retrotranscriptase enzyme) in this way I know if cDNA has a good integrity (a good solid 1 band of a HKG) and that there is no DNA contamination (no band in RT-).  


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#9 merlav

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Posted 01 August 2018 - 12:27 PM

If need to do a semiquantitative to a low expression gene, then  I would recommend running samples in a high resolution agarose like for example metaphor or running in a acrylamide gel.  


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#10 yobou

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Posted 01 August 2018 - 06:46 PM

do you have a protocol for PAGE separation of PCR products?



#11 merlav

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Posted 02 August 2018 - 04:43 AM

I don't have a protocol per se and haven't done in more than 15 years, but what I remember is  that the gel is prepare similar to the one for a WB with the difference of the buffer use.  Acrylamide/bis  30%,  37.5:1 at 10-12% in TBE (1x or 0.5x), the TEMED and APS don't change.  


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#12 OldCloner

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Posted 03 August 2018 - 04:35 AM

You can invest in some Metaphor agarose or just increasing the standard agarose % can help with the electrophoresis issues. I'd also recommend eliminating the variability of your running buffer batches by buying a concentrated 10 X TBE running buffer. It is already the proper pH and everything, just dilute it.  I preferred TAE myself, because you can buy that in a 50 X concentration,and it will not salt out (precipitate) on storage. And it is a good as TBE for most daily applications not involving high current. Buffer issues will then be a thing of the past!

 

BTW if you do buy Metaphor agarose be careful when you solubilize it, it boils over really easily.  I think it will dissolve easier in TAE than TBE, too.

 

Also, as merlav asked, what is the purpose of this end-point PCR from cDNA? Are you testing your assay before trying a qPCR?



#13 merlav

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Posted 03 August 2018 - 09:27 AM

Yes metaphor is trickier to prepare, it must let hydrate for 10 minutes on stirrer before heating. It must watch carefully because boils (and burn) easily. 


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie

#14 yobou

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Posted 03 August 2018 - 10:20 AM

purpose: conventional PCR of one of the ER stress markers: xbp1 cleavage.

I have acrylamide/bisacryl:29:1 ratio will it be suitable? shoould I use TBE buffer volume instead of the same water volume that is usually added when preparing PAGE? Should it be native PAGE? how about STACKING GEL in this case? 

any info is highly appreciated

thanks



#15 OldCloner

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Posted 03 August 2018 - 11:53 AM

Here's where you need to consult a standard lab manual. Does your lab have some version of Molecular Cloning, a Lab Manual from Cold Spring Harbor (author list varies with edition but includes Sambrook). Any edition would do. Or Current Protocols in Molecular Biology (the big red multi-volume tome). I know everyone is going digital these days but I hope research labs still have some copies of these around. All the basic "how tos" are in there. Check your library! Also the acrylamide manufacturer might have some standard recipes for standard applications on their website or product insert.

 

I seem to recall that when running PA gels for small DNA fragments, there was no stacking gel.  But I haven't run one in a long time, either. My most recent lab employer seemed to be opting for less toxic options.

 

Perfecting your gel may only prove you still need to optimize your PCR rxn. Do you want to clone the product or just prove it is there (presence/absence assay?)


Edited by OldCloner, 03 August 2018 - 11:56 AM.





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