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Another primer dimers problem - (Aug/17/2011 )

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yasamino on Thu Aug 18 22:44:27 2011 said:


almost a doctor on Thu Aug 18 10:33:03 2011 said:


Agree with everything Adrian said, your buffer concentration is wrong. Also, I think your annealing time is a bit too long, I rarely run for more than 30sec.

I have a new question too, what is the concentration of your dNTPs and primers? 0.75ul in a final volume of 20ul, but of what stock concentration??? If you are only getting primer dimer, your primer concentration might be too high, and your dNTPs too low.

Regarding your question about MgCl2, 1.5-2.5mM refers to the final concentration of MgCl2 on you PCR reaction. So as Adrian said, if you are adding 2ul of 25mM in a 20ul reaction, you have 2mM MgCl2. You'll need to add more, or less for it to be 2.5mM or 1.5mM.


Hello almost a doctor,
Great ideas..thanks
Although why is buffer concentration wrong? In the protocol that comes along with the Taq it says the buffer is 10X buffer ..hhmmmm...

Also, my dNTP concentration is 10mM. My primer concentration is I guess 100uM since I multiply the nmol by 10 and that is how much water I add to it. What do you think? Is too high?

Oh so I guess for MgCl2 I do not have to dilute it..I basically play around with how much I add to the 20ul reaction mix. Right?
How did you figure out how much to add for each MgCl2 concentration?

Overall thanks for everything!!!


1. Your buffer is 10x, and you have a 20ul reaction. So you would need 2ul not the 2.5ul you are adding.
2. Yes I do think that your primer concentration is too high.
You usually need a final concentration in your PCR reaction of approximately 0.2uM to 1.0uM, what you are adding is close to 4mM.
The concentration you have is for your STOCK solution for long term storage, which you should dilute to make your WORKING solution. If I have my working stock at 100uM, I would usually do a 1/10 dilution for working stock and then use about 0.5ul of that per reaction depending on the polymerase I'm using.

I would recommend downloading the product information for the specific polymerase you are using and then design your reaction from there. It will tell you the optimal concentrations and cycle temps and lengths for that polymerase.

-leelee-

A control reaction is a DNA extract that MUST work with your primers (usually a cloned copy of your gene or a DNA extract that gives a good signal). When doing PCR you should include one positiv and one negative control in each run - means you PCR your samples + the postive control that is expected to work + a negative control that must be negative (usually just the mastermix and water instead of the DNA). This allows you to control your PCRs - i.e. you do not need to worry why your results are negative when the postive control fails - this means something was wrong with your PCR. If the negative control is positive you cannot trust your results as well because you have a DNA contamination somewhere.

Of course this is difficult when you have new primers and do not know if they work or not - have you only ordered one set of primers or have you more primer pairs to test?

-gebirgsziege-

As it's already been said by leelee, you are using way too much primers. No wonder you get dimers ;) Dilute your stock to 10uM and then use 0.5-1ul for your final 20ul PCR reaction. Also, leelee already explained why your buffer concentration is wrong, is the final concentration in the PCR reaction that is wrong, not the 10x buffer.

585bp should not be too hard to amplify. I'd definitely try the settings Adrian said, your current Annealing and Extension times are too long. In fact I'd do 30sec at 94C, 30sec at 55C, 30sec at 72C for 30-35 cycles.

I personally would repeat a gradient PCR lowering the primer concentration, changing the cycling times, and adjusting the buffer concentration to be right. Also check you MgCl2 concentration, and what final concentration invitrogen suggest. Which also reminds me, make sure the 10x polymerase buffer you are using doesn't have any MgCl2 already.

IF that doesn't work, then is time to think about redesigning primers. To which I would add: TA clone your product and cut it from there, rather than digest the PCR product directly. But then that's personal preferences.

Oh! And I think your dNTPs concentration is fine but you might have to alter it if you change the MgCl2 concentration, but I wouldnt worry to much about that just now.

Good luck, and let us know if/when you get your product! :)

-almost a doctor-

Adrian K on Fri Aug 19 03:02:28 2011 said:


yasamino on Thu Aug 18 22:30:25 2011 said:


Adrian K on Thu Aug 18 05:39:17 2011 said:


Can I know your product length???
Is this a hoststart taq (platinum taq from invitrogen?)
Just to confirm, your final PCR volume is 20 or 25ul? This is because if your final volume is 20ul, your 10X taq buffer should be 2ul per reaction instead of 2.5ul.
Also, you are using 2.5mM MgCl2 here. Usually 2mM will be enough (1.6ul in a total 20ul reaction). This could be the possible reason for your reaction to fail...

And I not sure about formamide. I use DMSO, 5%. (In a total 20ul, you use 1ul DMSO.) hope this helps


Hello Adrian,

Thanks for all this.. you are very helpful.
In response to your questions, my product length is 585. The Taq that I am using is not platinum it just says Taq polymerase but it comes in a similar red box. My reaction mix is a total of 20ul. And I can't wait to try those hints and hope for the best!!
Thanks again!


Hmn, perhaps you a re using one of these:
http://tools.invitro...qrecomb_pps.pdf
http://tools.invitro...qnative_pps.pdf
http://tools.invitro...rimetaq_man.pdf
http://tools.invitro...ls/11508017.pdf

If thats the case, please look again your MgCl2 concentration: This is because all Mgcl2 supplies were in 50mM (by invitrogen) rather than 25mM that you mentioned, and you are ending up using double of the Mgcl2 concentrations. Or, you are using one of the old discontinued expired products?

I would suggest the following condition for your PCR: (to slightly shorten your time)
94C for 4min
94C for 45s
Anneal T for 30s
72C for 30s go to step 2 repeat 30 times
72C for 5min


Hello Adrian,

Great!!
I am sorry I mentioned that I used Invitrogen. Well I did before, but that specific run I used a bioshop kit which I buy from our science store. It is 25mM. I am sorry :D

I will take your steps of PCR cycles into consideration. This is awesome!
Thanks again!!

-yasamino-

leelee on Fri Aug 19 04:10:37 2011 said:


yasamino on Thu Aug 18 22:44:27 2011 said:


almost a doctor on Thu Aug 18 10:33:03 2011 said:


Agree with everything Adrian said, your buffer concentration is wrong. Also, I think your annealing time is a bit too long, I rarely run for more than 30sec.

I have a new question too, what is the concentration of your dNTPs and primers? 0.75ul in a final volume of 20ul, but of what stock concentration??? If you are only getting primer dimer, your primer concentration might be too high, and your dNTPs too low.

Regarding your question about MgCl2, 1.5-2.5mM refers to the final concentration of MgCl2 on you PCR reaction. So as Adrian said, if you are adding 2ul of 25mM in a 20ul reaction, you have 2mM MgCl2. You'll need to add more, or less for it to be 2.5mM or 1.5mM.


Hello almost a doctor,
Great ideas..thanks
Although why is buffer concentration wrong? In the protocol that comes along with the Taq it says the buffer is 10X buffer ..hhmmmm...

Also, my dNTP concentration is 10mM. My primer concentration is I guess 100uM since I multiply the nmol by 10 and that is how much water I add to it. What do you think? Is too high?

Oh so I guess for MgCl2 I do not have to dilute it..I basically play around with how much I add to the 20ul reaction mix. Right?
How did you figure out how much to add for each MgCl2 concentration?

Overall thanks for everything!!!


1. Your buffer is 10x, and you have a 20ul reaction. So you would need 2ul not the 2.5ul you are adding.
2. Yes I do think that your primer concentration is too high.
You usually need a final concentration in your PCR reaction of approximately 0.2uM to 1.0uM, what you are adding is close to 4mM.
The concentration you have is for your STOCK solution for long term storage, which you should dilute to make your WORKING solution. If I have my working stock at 100uM, I would usually do a 1/10 dilution for working stock and then use about 0.5ul of that per reaction depending on the polymerase I'm using.

I would recommend downloading the product information for the specific polymerase you are using and then design your reaction from there. It will tell you the optimal concentrations and cycle temps and lengths for that polymerase.



Hi Leelee,

Thanks for the clarification!!!
Wow I was off for my primer concentration hopefully that would lead be somewhere once corrected and everything.
I will definitely look at my protocol for my polymerase and like you said take into consideration what they say
Thanks dear :D:D

-yasamino-

gebirgsziege on Fri Aug 19 07:14:20 2011 said:


A control reaction is a DNA extract that MUST work with your primers (usually a cloned copy of your gene or a DNA extract that gives a good signal). When doing PCR you should include one positiv and one negative control in each run - means you PCR your samples + the postive control that is expected to work + a negative control that must be negative (usually just the mastermix and water instead of the DNA). This allows you to control your PCRs - i.e. you do not need to worry why your results are negative when the postive control fails - this means something was wrong with your PCR. If the negative control is positive you cannot trust your results as well because you have a DNA contamination somewhere.

Of course this is difficult when you have new primers and do not know if they work or not - have you only ordered one set of primers or have you more primer pairs to test?


Hello gebirgsziege,

Awesome thanks for the detailed explanation regarding controls. I am really fresh on this. I greatly appreciate your time in this.
It makes a lot of sense. I only have one set of primers but I will definitely order another set, my professor said the samethig too :D

Thanks dude!

-yasamino-

almost a doctor on Fri Aug 19 08:40:33 2011 said:


As it's already been said by leelee, you are using way too much primers. No wonder you get dimers Dilute your stock to 10uM and then use 0.5-1ul for your final 20ul PCR reaction. Also, leelee already explained why your buffer concentration is wrong, is the final concentration in the PCR reaction that is wrong, not the 10x buffer.

585bp should not be too hard to amplify. I'd definitely try the settings Adrian said, your current Annealing and Extension times are too long. In fact I'd do 30sec at 94C, 30sec at 55C, 30sec at 72C for 30-35 cycles.

I personally would repeat a gradient PCR lowering the primer concentration, changing the cycling times, and adjusting the buffer concentration to be right. Also check you MgCl2 concentration, and what final concentration invitrogen suggest. Which also reminds me, make sure the 10x polymerase buffer you are using doesn't have any MgCl2 already.

IF that doesn't work, then is time to think about redesigning primers. To which I would add: TA clone your product and cut it from there, rather than digest the PCR product directly. But then that's personal preferences.

Oh! And I think your dNTPs concentration is fine but you might have to alter it if you change the MgCl2 concentration, but I wouldnt worry to much about that just now.

Good luck, and let us know if/when you get your product!



Hi Almost a doctor,

Great!!

It really makes alot of sense to me now. I will try all the ways all of you mentioned and let you know if anything happens with me (cross your fingers hehe). Honestly you are very helpful and I will try everything mentioned since I did not have much knowledge about all the troubleshooting in regards to PCRs.

I will let my professor know about your idea regarding PCR product digestion and TA cloning for sure

Thanks again for everything!!!!

-yasamino-

yasamino on Sun Aug 21 04:09:45 2011 said:


Hello Adrian,

Great!!
I am sorry I mentioned that I used Invitrogen. Well I did before, but that specific run I used a bioshop kit which I buy from our science store. It is 25mM. I am sorry

I will take your steps of PCR cycles into consideration. This is awesome!
Thanks again!!


Dear yasamino,
Hahahaha... If you want people to help you accurately, please provide accurate information next time. No worries,no body will copy your PCR mastermix formula because almost all the people who responded to you here have their own mastermixes which worked for them...

Can you show your recent gel picture?

-Adrian K-

Hello All,

I have attached a gel image of the recent PCR I did using the following conditions:

10X buffer-2ul
25mM MgCl2-1.6ul
10mM dNTP-0.75ul
10uM Forward Primer-0.5ul
10uM Reverse Primer-0.5ul
Taq-0.3ul
cDNA template-4ul (for concentration check first post of mine)
dH2O-add up to 20ul

PCR conditios
94C-4min
94C-45sec
Anneal (I did a gradient of 52-62C)-30sec
72C-30sec go to 2 repeat 30 times
72C-5min
4-for ever

My product size is as mentioned earlier in posts 585 bp. The gel image has 6 products that are very faint and they correspond to the following temps

From left
1-53.7
2-54.8
3-56.3
4-58.0
5-59.4
6-60.5
Note: the first lane is a 1kb ladder, and between each one i skipped a lane.

So there are really faint bands, and 2 seems to be the brightest but looks like a smear. This sort of gives me hope before I did not see anything just primer dimers, but I obviously have to work more on it. Should I increase cDNA template? Is my cDNA contaminated or degraded or..? What can I do for even better results? I was thinking of trying 55 as annealing temp.?

Thanks ALL and I GREATLY APPRECIATE all the help!!!!!
YasaminoAttached Image

-yasamino-

Dear Yasamino,
There are six lanes, For band number 2 &3, there are 2 bands.
Maybe I am wrong, but I do not find that your band sizes were corresponded with the size you wanted. it is like 1.2kb and 1.8kb. Which brand of 1kb ladder you use, can provide the catalog number as well as a reference?

Let's say the band you wanted is the bottom band, I suggest you directly just pool all your samples, run it in agarose gel, do a gel purification and elution and finally do a cloning into your desired vector. There is no need to waste so much time on PCR optimization.

Is hard to say in this stage that whether your cDNA is degraded or whether you should increase it. I suggest you try to use your genomic DNA as control and see if there is any differences, provided your primer was not designed on the splicing site. Also, addition of Betaine or DMSO might help to eliminate the unspecificity binding and perhaps reduce smear. This is not the issue of primer dimmer.

-Adrian K-
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