When I work based on the protocol of my kit, I just see high probe peaks (blue peaks). My D fragments seems that doesn't exist and look like my PCR hasn't worked. Is this opinion true or not? Can anybody answer to my question?
The answer may depend on the number of splice variants and/or gene deletions you may be dealing with in your sample type, but given the paucity of information in your original post, one cannot say for sure; not enough background information is provided.
Your question seems to be controversial: first you say that you have high blue peaks from the probes, than you state that you have problems with pcr (D fragments). I agree with Jack M.Gallup, please specify better your question.
Best Regards
my D fragments are very low and seems that my initial denaturation step is ok, but i have a long 88nt fragment in all of my samples that is higher than 92nt fragment ( almost higher than of 1/4 height of 92nt fragment). i don't know ligation worked or not.
Generally it's better to dilute DNA in TE buffer and not water. The pH of the solution should be kept over 7.5, slightly basic.
The final volume of the DNA should be 5 ul......with the addiction of SALSA buffer and probemix (3ul) it becomes 8ul, for the overnight hybridisation.
This is the MRC Holland protocol and general suggestions.
With this protocol I never had problems, also with DNA samples referred to me from other centers and extracted with different protocols (and not always very clean).
Have a look also on the calibration and precision of the thermal cycler.
Good Luck
Alessandro
dear Alessandro
so thanks for your respond. if it's possible can i send some of my results( raw data) to you and give your opinion about them to me.
I wrote back to Jamila Iqbal some suggestions. Check them with your current protocol, eventually send me a couple of printscreens of your electropherograms.
I have received the MLPA results from A'zam Azimi as well.
I would love to help, but I really don't understand what the question is and am having problems communicating this.
@A'zam: The data itself seems alright, but I need more info to tell fully. I think the sizing may be going wrong, so maybe what you are seeing as 88 nt may actually be the 92nt peak you are expecting. I ask you again: More input please! Which locus is this, and what are the details to the assay? (Number of peaks for each fluorescent dye etc.)
I don't know if this topic is still active, but I have to say that impurity of the input DNA, and low, as much as high amount of it, damaged, salted... may cause imbalance of the control and other probe fragments. I must say, in our experience, if you have DNA concentration below 20ng/ul you may start your denaturation with 6, 7, 8, even 9ul of DNA, it works fine. Then, if you have done everything according to standard MLPA recommendations, be careful to work with fresh chemicals (polymer, formamide etc) and with right (optimized) capillary electrophoresis settings. You may analyze your samples using Coffalyser.NET, but you may also use GeneMapper (or GeneMarker) + Excel sheets from NGRL, or + RH-MLPA-Analysis software. If the problem is still exists contact [email protected]. All of you have a lot of experience in performing MLPA, but for the "new users", I attached some "old" troubleshooting files. I hope that will help.
i actually sent A'zam Azimi some analyzed files on Tuesday. All his MLPAs worked fine - I haven't heard from him since though.
so thanks of your help and suggestions. excuse me if i didn't see your respond.
dear SANJA
did you have worked with HBB p102 kit for diagnosis? I have one same sample patient with two different results , one completly normal and another have duplications in some of its probes? this sample was extracted and diluted in two different way, one diluted in dH2O with final concentration 22ng ( i used 5microliter of this sample in my MLPA denaturation step), another without any dilution because of its low primary concentration (6ng).. i used same volume of this sample such as first samplt (5 microliter). these two samples showed different results, first sample had normal copy number and second had duplications in some probes. which result is acceptable for confident diagnosis?
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