9 replies to this topic

[kyakhoob]

Hello ...
I m working with five sets of ssr primer which i have tested earlier and i got bands at 300 bp. but now  i am using same set of primers,  same reaction mixture and same dna but i m getting faint bands at 50 bp..please help..please

[hobglobin]

Sounds as if this are primer-dimers that occur at this size....i.e. your PCR did not work...perhaps one of the reagents is kaput now (often dNTPs), or DNA degraded, or you forgot to add a reagent to the reaction (Taq, dNTPs, ...)?  You have to try out and use a positive control, then you know that the reagents are okay.

[kyakhoob]

habglobin

thank you for your suggestion actually i have tried everything. i have added every reagent properly..i tested the DNA and found OK..primers are few month old. my guide told me that the primers might get contaminated. is there any way to know whether the primers are okay or not. please help.

[hobglobin]

kyakhoob, on 28 September 2012 - 04:03 AM, said:

habglobin

thank you for your suggestion actually i have tried everything. i have added every reagent properly..i tested the DNA and found OK..primers are few month old. my guide told me that the primers might get contaminated. is there any way to know whether the primers are okay or not. please help.

how did you check the DNA then? and primers, I'd also replace, i.e. throw away the working solutions and thawing new ones (my primer working solutions are frozen as several diluted aliquots from the stock).
also checking the thermal cycler is an idea, i.e. asking if colleagues have no problems with their PCRs and if your program is still untouched...

[kyakhoob]

Habglobin
sorry for late reply.
Thank you for your suggestion. I checked the DNA by running genomic DNA in 0.8% gel. I checked the thermocycler and it is OK. Can I centrifuge the the working primer solution before  preparing the master mix to avoid thawing of primers....???? please. help

[kyakhoob]

hobglobin, on 28 September 2012 - 05:41 AM, said:

kyakhoob, on 28 September 2012 - 04:03 AM, said:

habglobin

thank you for your suggestion actually i have tried everything. i have added every reagent properly..i tested the DNA and found OK..primers are few month old. my guide told me that the primers might get contaminated. is there any way to know whether the primers are okay or not. please help.

how did you check the DNA then? and primers, I'd also replace, i.e. throw away the working solutions and thawing new ones (my primer working solutions are frozen as several diluted aliquots from the stock).
also checking the thermal cycler is an idea, i.e. asking if colleagues have no problems with their PCRs and if your program is still untouched...

Habglobin
sorry for late reply.
Thank you for your suggestion. I checked the DNA by running genomic DNA in 0.8% gel. I checked the thermocycler and it is OK. Can I centrifuge the the working primer solution before  preparing themaster mix to avoid thawing of primers....???? please. help

[hobglobin]

and the DNA was ok? and I didn't get what centrifuging primer working solution has to do with thawing? I store my working solutions in the fridge and centrifuge them shortly before using, to get down the condensed water and mix it. The same with other solutions from the fridge where this problem may occur.

[kyakhoob]

hobglobin, on 09 October 2012 - 07:52 AM, said:

and the DNA was ok? and I didn't get what centrifuging primer working solution has to do with thawing? I store my working solutions in the fridge and centrifuge them shortly before using, to get down the condensed water and mix it. The same with other solutions from the fridge where this problem may occur.

'hobglobin
yes my DNA is ok. As you said that that "your primer working solutions are frozen as several diluted aliquots from the stock. as, you store  your working solutions in the fridge and centrifuge them shortly before using, to get down the condensed water and mix it. " my question was that should I centrifuge all solution (Buffer, MgCl2, dNTP) including my primer working solution????
I used to centrifuge only the MgCl2 before using it. and i use freshly prepared dNTP each time. Is it important to centrifuge the primer working solution before using it???
please suggest me.

[hobglobin]

well I actually do it when they were quite long in the fridge, because when using them frequently, the condensing water is no problem...anyway with frozen and then thawed solutions mixing is more important to avoid heterogeneous concentrations within the tube...especially with MgCl₂ and buffer....and after this, the centrifuging is not necessary of course.

[kyakhoob]

hobglobin, on 10 October 2012 - 08:32 AM, said:

well I actually do it when they were quite long in the fridge, because when using them frequently, the condensing water is no problem...anyway with frozen and then thawed solutions mixing is more important to avoid heterogeneous concentrations within the tube...especially with MgCl₂ and buffer....and after this, the centrifuging is not necessary of course.

Ok..I am going to try it again..... fingers crossed ...