what may have gone wrong? - (Jun/05/2018 )

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.  

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?

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. 

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

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?)

You will need to explain more of what is the work about to get the right help.   A conventional PCR usually is using DNA as template.  

Yes it will be suitable the acryl/bis. TBE 1x or 0.5X volume  same as replacing the water and resolving buffer for the total volume of the gel.  No stacking gel is needed. For example total volume needed to do the gel is 15 mL, then the volume of TBE=15mL-acry/bis mix-TEMED-APS.  Remember to add the buffer in the same way that if running a WB we don't want burnt chambers. 

the cleaved band of xbp-1 is adjacent to the uncleaved xbp-1. hopefully PAGE may be helpful.

So, I read up a little on X-box binding protein (XBP1) and it has mRNAs that can be spliced or un-spliced, is that it?  So you could actually expect PCR bands of two different sizes, if your primers are on opposite sides of the intron? Is that what we are talking about? We are not sure exactly what you expect to see on your gel. And why you are not doing qPCR. As merlav said, some better information might help.

here is the source paper that I used its primer for XBP-1. typical results are shown http://jpet.aspetjournals.org/content/311/1/388.long

What I maybe missed in all that is what is the expected size od cleaved and uncleaved? You mentioned 150 and 270+ bands, is it the expected size? Or rather expected size with a double band (if so, what is the bp difference between the double band).

So is the ONLY problem there the diffuse bands?

In paper mentioned, I so only one gell picture and it's pretty crappy if you ask me, it would need much better resolving and misses a marker completely. So reason why theirs is not diffuse is they didn't run it such long.

For samples with very short fragments (150 would count in that, especially if they are after restriction) I found better to use SB buffer. It can run on much higher voltage (at least 1/3 higher) and thus the small band do not diffuse that much. One catch though, it doesn't work well with high-salt samples, if you put restriction reaction right on gel, it would get distorted and need to be desalted before running on gel (add 2.5x volume of 100% ethanol, overnigh on -20 C (or hour on -80 C) spin 10 min/12 000 g/ 4 C and disolve in appropriate ammount of MiliQ water). I did several restrictions with a very small band with this, worked nicely.

Generally runing on higher voltage/shorter time will make bands less diffuse, but buffers don't like higher voltages and warm too much and conduct worse. SB is a better conductor.