Jump to content

  • Log in with Facebook Log in with Twitter Log in with Windows Live Log In with Google      Sign In   
  • Create Account

Submit your paper to J Biol Methods today!
Photo
- - - - -

what may have gone wrong?


  • Please log in to reply
21 replies to this topic

#16 merlav

merlav

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 111 posts
2
Neutral

Posted 03 August 2018 - 11:57 AM

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. 


Edited by merlav, 03 August 2018 - 12:03 PM.

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

#17 yobou

yobou

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 102 posts
1
Neutral

Posted 03 August 2018 - 12:37 PM

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



#18 OldCloner

OldCloner

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
5
Neutral

Posted 06 August 2018 - 05:40 AM

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.



#19 yobou

yobou

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 102 posts
1
Neutral

Posted 06 August 2018 - 07:11 AM

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


Edited by yobou, 06 August 2018 - 07:12 AM.


#20 Trof

Trof

    Brain on a stick

  • Global Moderators
  • PipPipPipPipPipPipPipPipPipPip
  • 1,369 posts
135
Excellent

Posted 06 August 2018 - 07:55 AM

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.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon


#21 OldCloner

OldCloner

    Enthusiast

  • Active Members
  • PipPipPipPipPip
  • 52 posts
5
Neutral

Posted 06 August 2018 - 10:34 AM

Hmm- I agree with trof: that is a dodgy looking gel in Fig. 5c; there is so little separation between the bands that you could account for the change from left to right by saying the gel was smiling. Also, my problem with this is that they are trying to make quantitative judgements at an endpoint. It is quite possible for both spliced and un-spliced cDNAs to amplify to plateau in an endpoint PCR no matter how different the amount of starting material.  If it was me I‘d design 2 sets of primers to distinguish the two forms, and do qPCR.



#22 merlav

merlav

    Veteran

  • Active Members
  • PipPipPipPipPipPipPipPipPipPip
  • 111 posts
2
Neutral

Posted 06 August 2018 - 11:43 AM

I had done end point PCR to a cDNA for the purpose to detect the loss of imprinting of a gene.  When I received the job to continue that project the first thing that I did was changing the primers so the result of the enzyme cleavage would be an asymmetric cut with a very distinctive  pattern that there was no doubt about the results..  After some time was able to convince the boss that we can do a better assay with a QPCR and I designed 2 probes each with a different fluorophores to detect the cut/uncut template. 

 

If possible change the assay to a QPCR either designing 2 primer set that can distinguish between both forms as OldCloner told, good part sybr green is cheaper downside you have to assays for each primer set apart.    The other way to do the QPCR is using one primer set and 2 probes (make sure fluorophores don't overlap), the good part of using  probes its that no matter if you have either form or both forms at the same time you will be able to detect it without doubts in a single reaction so you will save in enzyme and time. QPCR may be a bit pricier, but much pricier is repeating assays that don't give a clear result and  more pricier is a rejected paper because results aren't clear for the reviewers. 


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




Home - About - Terms of Service - Privacy - Contact Us

©1999-2013 Protocol Online, All rights reserved.