Mrenrisco

I need some tips for my PCR

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Hi guys,

so I started my PCR last week, I'm analysing capsicum species using RAPD markers, but I'm a little confuse of how should I procede with my PCR.

First of all, my professor said that I couldn't make the mix all at once and then distribute to micro tubes with my DNA samples, she said that I should prepare the mix separately. Well that's quite annoying, because I have a lot of samples and I must amplify each primer 3 times. Is it really necessary prepare the mix for each sample?

Second, for my project my professor selected 20 primers to analyze the polimorphism and etc. will each primer have its own temperature of annealing, desnaturation etc. or should I configure my termocycler once and use the same program for all primers?

and last but not least, this was my first electrophoresys of a PCR:

image.png.80059a0f419c30f959a38c107d628ba5.png

this was just a test, I used 11 samples: some of them amplified, some of them not... but I'm not actually worried about that, I want to know why those wich amplified didn't show clear bands, and why my blank (the sample right before the ladder) stained.

 

 

Oh, I used the primer OPA 020 for this first test

Edited by Mrenrisco

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On 7/11/2019 at 10:11 PM, Mrenrisco said:

Hi guys,

so I started my PCR last week, I'm analysing capsicum species using RAPD markers, but I'm a little confuse of how should I procede with my PCR.

First of all, my professor said that I couldn't make the mix all at once and then distribute to micro tubes with my DNA samples, she said that I should prepare the mix separately. Well that's quite annoying, because I have a lot of samples and I must amplify each primer 3 times. Is it really necessary prepare the mix for each sample?

Second, for my project my professor selected 20 primers to analyze the polimorphism and etc. will each primer have its own temperature of annealing, desnaturation etc. or should I configure my termocycler once and use the same program for all primers?

and last but not least, this was my first electrophoresys of a PCR:

image.png.80059a0f419c30f959a38c107d628ba5.png

this was just a test, I used 11 samples: some of them amplified, some of them not... but I'm not actually worried about that, I want to know why those wich amplified didn't show clear bands, and why my blank (the sample right before the ladder) stained.

 

 

Oh, I used the primer OPA 020 for this first test

Thanks for your question. Just as this is your first gel electrophoresis of PCR, this is my first time reflecting on PCR in quite a few years, so don't take my word for it completely but I'm going based on memory here to try to help you/answer your question since I see no one else has yet. Hopefully someone else chimes in and corrects me where I'm wrong.

 

1.- The impression I'm getting from your professor, since she's asking you to do them separately, is that she wants you to retain how to make the mix and also she wants you to be careful/not wasteful of the products.

It's a tedious process, and the best way to learn is by repetition. The fact that your professor's asking you to repetitively go through the protocol shows me she really wants you to learn and perfect your method of PCR.

More importantly, I think your professor doesn't want you to affect the quality of the outcome of PCR. Perhaps making a larger batch could cause you to dilute the primers more than what they should be, which with one mistake the whole batch would be ruined. From what I recall (years ago), primers are expensive so she also probably doesn't want you to waste the whole thing in one go.

Instead, if you work on them one at a time, it'll help for keeping the outcome consistent with minimal/negligent errors. So, although I don't think it's a necessary measure, I do feel it would be the smarter thing to do if you really want to ingrain PCR technique and also take precautionary measures as well as keep the outcome consistent.

Also, it could be the case that one of the primers may require a different step which cannot be applied to other primers.

 

2.- Primer = DNA piece that will be basically copied/recreated. DNA is molecular compound which means having the DNA of one species of bell pepper should be similar to another species of bell pepper. This means the environment in which DNA reproduces should be the same. So, no I don't believe each primer will have temperatures different than one another's but perhaps there may be additional steps required to produce a certain result you're looking for (one of the species).

 

3.- Blank is supposed to stain so that you have an independent/standard component to compare the rest of your strands with. The ones that didn't show either weren't dyed or something may have gone wrong – most probably a forgotten ingredient (which is probably the reason why your professor wants you to repetitively make the batches), or something may have gone wrong.

This link may help with that: https://www.fws.gov/aah/PDF/PCRTS1_NoBand.pdf

Try troubleshooting using that.

 

Hope this helps!

 

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Generally, as far as I see it, usage of materials is reduced when making stock solutions (especially when using enzyme which is quite sticky for instance) as there will always stick a little to your pipette tip. (of course, don't mix the different primers, but primer pair #1 with 3 samples can be done with 1 mix >>> add DNA/water to tube, add pre-made primer/enzyme/buffer mix).

What TMX is saying regarding consistency; I have to disagree, using separate mixes when trying to compare samples is a way of introducing errors, as you do not pipette accurately all the time.

Regarding primers, yes all primer pairs will have a optimal Tm and should be calculated, the temperatures you will use in your PCR will be based on the enzyme you use and the Tm. 
When you see non-specific bands, you may want to consider optimization using additional DMSO, different MgCl2 concentrations, usage of a different buffer (in case your primer has a high (> 60-70%) GC content), or usage of a different enzyme. 

Primers are (in my opinion) not very expensive, when comparing with the enzymes used.

Reasons for not seeing bands could also be due to bad (c)DNA quality (although I personally think it is a bit unlikely) or non-working primers (but if they work for some samples and not for others, than that isn't the problem). 

I personally would go about it in a primer-by-primer kind of method; find the optimal conditions for each primer pair and note them down (both from Tm calculations, DMSO/MgCl2 concentrations for GC content and product length, and from experimental evidence seen in your gels) then once one works perfectly, do it for all of them. Yes it is a lot of work, but I suppose with 20 primers, it is quite a big project anyway.

Please take a look at PCR troubleshooting, and of course try to understand why certain chemicals effect your results and how the different enzymes work (sometimes changing enzyme can solve issues and personally I sometimes don't understand WHY this solves the issue, but it does). https://international.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide (or any other troubleshooting guide, this one is just the first one that came up on google).

Hope this helps, please discuss with your professor as they will be more knowledgeable and may disagree (while I have done my fair share of PCR, I wouldn't consider myself an expert).

Goodluck!

-Dagl

 

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