How to eliminate primer dimer in MSP - (Jan/04/2006 )
Hi,pcrman .
I am doing the MSP at cell lines.I can get the band that i want,but the primer dimers is sevesely.The primer concentration is 0.4uM.I could not delete it by any way.Could you tell me what should I do to deliminate the primer dimer.Thanks a lot.!
MSPnewer
400nM primer concentration is quite high.
you could try reducing this to 200nM (this is the concentration I routinely use for PCR)
Nick
you could try reducing this to 200nM (this is the concentration I routinely use for PCR)
Nick
Hi,nick.Thank you very much!I reduced the primer concentration to 200 nM,and it work well.The primer dimers is disappear.However it came out the smear and the band is very veil.My sDNA is 100ng !What are the factors for the veil band?and how does the smear arise?Thank you very much!!
MSPnewer
MSPnewer,
I am not too sure what you mean by veil bands.....can you post an image of your gel to the forum so we can all see??
Nick
Hi,Nick
I mean the band is faint . Now I runinto some other problems.I had ever get my beautiful M and U bands two weeks ago!However,i couldnot get anything except the primer dimers from then.I find it unstabel to do MSPCR. There are many influences especially the template after modification. Some of my classmates said that primers with high GC% are easilly degraded.Maybe my primers that i design from the Methprimer are useless!
Do you think that my primer work for i have get my bands?
Now i donot what to do !Could you help me,please?Thank you very much !
MSPnewer
there could be a number of issues, the bands being faint could mean that you have very little starting material.
are you able to post an example gel image and your MSP primer sequences for us all to have a look??
Nick
are you able to post an example gel image and your MSP primer sequences for us all to have a look??
Nick
Hi Nick
I post some of my gel image.I am sorry they are not clear.Could you point out what is the isseue.I use 2.0ug gDNA.After the modification,i could see nothing.The OD260 is very low,usually 0.0050~0.0090,sometimes i get negative value,such as -0.0115.I use the Promega Wizard DNA Clean-Up System. I think i have lost the DNA.
Another question is that how long could I storge my primers in -20℃ refrigerator.Maybe my primers have been degraded.
Looking forward to your answers,nick.Thanks a lot.
MSPnewer.
hmmmm very perplexing,
I was wondering, how were the primers designed? with methprimer? or were they from a published paper? The primer pairs could have been designed as if they were normal primer pairs in which case they would not work properly. They need to be strand specific. Also, how were the primers purified? with MSP sets I usually get at least cartridge purification, or if you can afford it, PAGE as the freyed 3'ends will result in no amplification.
I would check to see if your primers were efficently designed and it may well be that primer dimers form at the temps for your PCRs which would mean you would hav to redesign them.
it's interesting to see that reducing the primer conc loses the band you are after.
Bisulfite DNA if you are performing it with conventional methods, is very destructive, if you start with 2ug of gDNA you end up with maybe 200ng or less. You don't really have to spec it as it contains uracil and will give a funny A260 reading anyway.
My primer stocks last for upto 1 year in the freezer -20C they are resuspended in TE at 250uM concentration for long term stability.
Nick
I was wondering, how were the primers designed? with methprimer? or were they from a published paper? The primer pairs could have been designed as if they were normal primer pairs in which case they would not work properly. They need to be strand specific. Also, how were the primers purified? with MSP sets I usually get at least cartridge purification, or if you can afford it, PAGE as the freyed 3'ends will result in no amplification.
I would check to see if your primers were efficently designed and it may well be that primer dimers form at the temps for your PCRs which would mean you would hav to redesign them.
it's interesting to see that reducing the primer conc loses the band you are after.
Bisulfite DNA if you are performing it with conventional methods, is very destructive, if you start with 2ug of gDNA you end up with maybe 200ng or less. You don't really have to spec it as it contains uracil and will give a funny A260 reading anyway.
My primer stocks last for upto 1 year in the freezer -20C they are resuspended in TE at 250uM concentration for long term stability.
Nick
Hi,Nick.Thank you for your quick answer.
I designed my primers with Methprimer and they have ever work ,getting the bands that I want though I have not send them to sequence.What is wrong do you think with my primer?I have ever heard my classmates that primers designed from Methprimer work well.
MSPnewer
Hi, Nick,you said" Also, how were the primers purified? with MSP sets I usually get at least cartridge purification, or if you can afford it, PAGE as the freyed 3'ends will result in no amplification."
I order primers from the company directly,do you think I should purify it again?
And you think" You don't really have to spec it as it contains uracil and will give a funny A260 reading anyway."So you think it is no use to determine the concentration at all?
Thanks sincerely!
Lisa