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General PCR discussion

PCR RT-PCR Primers

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#1 That_Lab_Guy

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Posted 30 October 2013 - 06:30 PM

Good day to everyone out there in the Bio community.

 

I am relatively new in the lab as I just started working in the lab after completing my biomedical degree and there are some tips I would like to learn from everyone and anyone out there who is willing to share any useful lab techniques and/or clarification of doubts.  As some of the lab work and equipment I have used during my undergraduate days are pretty much outdated, I would like to update myself with the latest information and methodologies in these lab techniques to better upgrade myself in today's ever changing world.

 

A brief introduction about myself before I start off.  I just graduated from the University of Bradford with a honours degree in Biomedical Science, specialising in the field of Medical Cell Biology.  I love sports and doing lab work.  I am currently working in a research lab dealing with exosomes.

 

And now on to my question.

I just ordered some primers from a primer design company for a RT PCR to be done in the next few days.  Is it common practice to mix the forward and reverse primers in order to facilitate the PCR work?  I mixed 10uL of the forward primer with the 10uL of the reverse primer and top up the mixture to 100uL by adding 80uL of UltraPure distilled water before putting them in the 4 deg Cel refrigerator.  Would like to find out if there's any points to take note regarding this and if I have done anything wrong.

 

Thank you.biggrin.png


Edited by That_Lab_Guy, 30 October 2013 - 06:31 PM.


#2 phage434

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Posted 30 October 2013 - 08:00 PM

That sounds fine, but limits you to using just that pair of primers. If you want to use a single primer, it is now difficult. Single primers can be paired with a different primer, or can be used for sequencing a PCR product.

 

I would also use TE rather than water for diluting primers, but there is some controversy about this position.



#3 That_Lab_Guy

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Posted 31 October 2013 - 12:16 AM

That sounds fine, but limits you to using just that pair of primers. If you want to use a single primer, it is now difficult. Single primers can be paired with a different primer, or can be used for sequencing a PCR product.

 

I would also use TE rather than water for diluting primers, but there is some controversy about this position.

 

Thanks for your reply phage434.  I forgot to add that I was trying to use these primers to test for the presence of PGK to determine if it's a normal cmyc gene or a viral cmyc gene.  I used the "Oligo6" software to determine my primer sequence.  The methodology is to use these primers to isolate the specific region on the gene sequence where PGK may or may not be present just before the cmyc gene in order to find out the origin of cmyc.  However, prior to that, I also intend to test if these pair of primers work before carrying out my experiment.



#4 Trof

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Posted 31 October 2013 - 01:17 AM

If you don't need primers separately too (as for sequencing with them) it's more convenient to mix them together. But I dilute my primers in 10mM Tris pH8 instead of water and some members here would swear for TE buffer for storage, especially longer one.

 

Rarely, you may observe worse amplification with the primer mix than when using them separately, that is due to binding at low temparatures, this can be corrected by quick denaturation and cooling down of the primer mix, before you add it to reaction. But that happend to me like.. once (and it was primer-probes mix) and I mixed like hundred of them already.


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#5 dpo

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Posted 31 October 2013 - 02:20 AM

Rarely, you may observe worse amplification with the primer mix than when using them separately, that is due to binding at low temparatures, this can be corrected by quick denaturation and cooling down of the primer mix, before you add it to reaction. But that happend to me like.. once (and it was primer-probes mix) and I mixed like hundred of them already.

 

@Trof: do you have any idea how this is possible? If you use the primer mix in a PCR, your first step is also a denaturation step so any primer dimers made in the primer mix would be expected to be denatured at that time, no? I'm asking because we recently ran into problems with one of our PCRs. When we make a primer mix of our 4 primers (it's a duplex reaction), the PCR doesn't work, but when we add them separately, it does work. The first time we tried out the primer mix (not freshly made but after freezing it once), it worked, but following experiments always failed, even when using a different aliqout of the primer mix. I must say it is a difficult template (GC rich) so we need DMSO in the reaction as well, but this is only added during PCR setup.

 

It would be interesting to try to denature the the primer mix before adding them, but we don't have a heating block in the room where we set up our PCRs and using one in another room is not possible (I work in a diagnostic setting with strict room specific functions).



#6 That_Lab_Guy

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Posted 31 October 2013 - 03:14 AM

 

Rarely, you may observe worse amplification with the primer mix than when using them separately, that is due to binding at low temparatures, this can be corrected by quick denaturation and cooling down of the primer mix, before you add it to reaction. But that happend to me like.. once (and it was primer-probes mix) and I mixed like hundred of them already.

 

@Trof: do you have any idea how this is possible? If you use the primer mix in a PCR, your first step is also a denaturation step so any primer dimers made in the primer mix would be expected to be denatured at that time, no? I'm asking because we recently ran into problems with one of our PCRs. When we make a primer mix of our 4 primers (it's a duplex reaction), the PCR doesn't work, but when we add them separately, it does work. The first time we tried out the primer mix (not freshly made but after freezing it once), it worked, but following experiments always failed, even when using a different aliqout of the primer mix. I must say it is a difficult template (GC rich) so we need DMSO in the reaction as well, but this is only added during PCR setup.

 

It would be interesting to try to denature the the primer mix before adding them, but we don't have a heating block in the room where we set up our PCRs and using one in another room is not possible (I work in a diagnostic setting with strict room specific functions).

 

 

I think it could be due to the primer design.  Too high annealing temperatures or too large negative ΔG values, giving rise to huge reduction in product yield as a result of formation of primer secondary structures.  Having said that, it becomes a headache when attempting to design primers.  I haven't actually tried denaturing the primer mix before adding them as I haven't done a duplex reaction as of yet.  But yeah, like you said it would be interesting to do so.  Also, is there really a need to add DMSO in PCR? Thank you.



#7 Trof

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Posted 31 October 2013 - 04:08 AM

@Trof: do you have any idea how this is possible? If you use the primer mix in a PCR, your first step is also a denaturation step so any primer dimers made in the primer mix would be expected to be denatured at that time, no? I'm asking because we recently ran into problems with one of our PCRs. When we make a primer mix of our 4 primers (it's a duplex reaction), the PCR doesn't work, but when we add them separately, it does work. The first time we tried out the primer mix (not freshly made but after freezing it once), it worked, but following experiments always failed, even when using a different aliqout of the primer mix. I must say it is a difficult template (GC rich) so we need DMSO in the reaction as well, but this is only added during PCR setup.

 

I think I've seen this as an offcial Roche recommendation in their protocols using primer-probe mix, there is a note there "Due to possible primer/primer interactions that occur during storage it may be necessary to preheat PCR primer-probe mix for 1 min 95 C before starting the reaction. This extra step will ensure optimum sensitivity."

I don't really searched for the reason, but I already encountered this situation once.
I may just guess that maybe the behaviour of primers is different in the final reaction mixture, so they maybe less willing to get out of any structure they created in storage. 

 

 

 

 

 

 

 

I think it could be due to the primer design.  Too high annealing temperatures or too large negative ΔG values, giving rise to huge reduction in product yield as a result of formation of primer secondary structures.  Having said that, it becomes a headache when attempting to design primers.  I haven't actually tried denaturing the primer mix before adding them as I haven't done a duplex reaction as of yet.  But yeah, like you said it would be interesting to do so.  Also, is there really a need to add DMSO in PCR? Thank you.

Well the problem is those primers don't form any undesirable structure when added separately, but some probably do upon a combined storage. This in not easily explained or predicted in the primer design, and it won't be unless there is an exact knowledge of what lies behind this, given that it is not very common thing.


About  that primer headache, you probably need to see some of the "bad" primers in action (like, some published ones, ones you "inherited") to have less headaches about deltaG values that softwares spit out wink.png
And I just design all my primers to slightly higher annealing tempreratures (60 C) because it increases specificity.

DMSO is a PCR additive used for decreasing the annealing temperature needed for specific binding, also it relaxes secondary structures on target DNA so it's commonly used for GC-rich templates. On the other hand they decrease activity of polymerase significantly with and increasing concentration.
So no you don't need it in normal PCR, but you do on GC-rich one, that wouldn't amplify otherwise (5% DMSO is currently only optimization I do, either if reaction doesn't work, or I can guess from the look at the amplified sequence that it might not, but I have a hell of a PCR Master mix wink.png ).


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


#8 phage434

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Posted 31 October 2013 - 04:22 AM

1 M betaine, 3-6%, in my experience, works better than DMSO for GC rich reactons. This is the same as "Q-solution."



#9 Trof

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Posted 31 October 2013 - 05:35 AM

I have to order it at last :) We have huge stock of injection-grade DMSO that went too off for clinical use and no-one want's to buy anything else now :)


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






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