23 replies to this topic

[Fluffy]

I performed a PCR for my gene and I have been getting primer dimers. I tried a temperature gradient two times, once a 58-68 gradient and another is a 45-55 gradient. Both times I got primer dimers only and no PCR product. To refer to my PCR details in terms of conditions look below.
I noticed you mentioned on this forum that one way is to titrate MgCl2 to 1.5-2.5mM final concentration gradient. Do you mean dilute it? And to dilute do I use H2O, please show me an example? And based on my original protocol, the initial MgCl2 concentration is 25mM and I added 2ul per reaction, would I add the same amount even if diluted to as low as 1.5-2.5mM?
You also mentioned to add formamide or DMSO. Formamide I have in lab is minimum 99.5% (100ml), to dilute to a final concentration of 2.5% do I use water? And is there a special way of adding it to the PCR reaction? How much should I add per reaction (refer to below for my reaction components), and do I still add up to 20ul including formamide?

PCR DETAILS
Primers (in brackets are restrictions sites)
Forward Primer (NdeI)
5’ (c a t a t g) a t g g t g g a c t t g g c t a g g g t 3’
Reverse Primer (XhoI)
5’ (c t c g a g) t t a g c t g g a c a a c a g c t t t t c a 3’
Forward: %GC: 55, Size: 20 bases, Tm: 60.77C
Reverse: %GC: 40.91, Size: 22bases, Tm: 60.05C

My PCR components:
10X Taq Buffer (invitrogen) 2.5ul per reaction
25mM MgCl2 (invitrogen) 2ul per reaction
dNTP 0.75ul per reaction
Forward 0.75ul per reaction
Reverse 0.75ul per reaction
Template 2 or 4ul per reaction (400ng/ul is template concentration)
Taq polymerase (invitrogen-500units/ul) 0.3ul per reaction
DEPC H2O add up to 20ul

My PCR conditions:
94C for 3min
94C for 45s
Anneal T for 1min
72C for 1min30s go to step 2 repeat 27 times
72C for 10min

I really hope you can help with this! I have been trying to find hints to clear things up for myself and it seems that you have a lot of them.
Thanks
Yasamino

[Adrian K]

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

[almost a doctor]

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.

[gebirgsziege]

are you using primers that worked before in your lab (and if did they work in your hands)? Or are you using newly designed primers or primers you picked from a paper etc. and do not know if these primers work on your template?
How does your control reaction look like - if the primers worked before you should use a sample that was fine before to test your reaction everytime you run a pcr....

[phage434]

In addition to all of the above issues, you will likely have problems at your next step, which I assume is to cut with NdeI and XhoI. For these enzymes to cut, you need to have six or so 5' additional bases (doesn't much matter what they are) on each primer.

You might be able to TA clone with these primers and then cut the product from the clone, but this is probably not what you had in mind.

I strongly recommend that if you are new to doing PCR reactions (and even later) that a commercial master mix leads to consistent results with far fewer opportunities to make mistakes. It also reduces the number of opportunities for contamination.

[Fluffy]

Adrian K, on 17 August 2011 - 09:39 PM, 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!

[Fluffy]

almost a doctor, on 18 August 2011 - 02:33 AM, 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!!!

[Fluffy]

gebirgsziege, on 18 August 2011 - 03:33 AM, said:

are you using primers that worked before in your lab (and if did they work in your hands)? Or are you using newly designed primers or primers you picked from a paper etc. and do not know if these primers work on your template?
How does your control reaction look like - if the primers worked before you should use a sample that was fine before to test your reaction everytime you run a pcr....

Hello gebirgsziege,
The primer I designed, have not been tested before. And honestly I dont have a control reaction, what do controls have to be like in general?

Thanks :D

[Fluffy]

phage434, on 18 August 2011 - 04:42 AM, said:

In addition to all of the above issues, you will likely have problems at your next step, which I assume is to cut with NdeI and XhoI. For these enzymes to cut, you need to have six or so 5' additional bases (doesn't much matter what they are) on each primer.

You might be able to TA clone with these primers and then cut the product from the clone, but this is probably not what you had in mind.

I strongly recommend that if you are new to doing PCR reactions (and even later) that a commercial master mix leads to consistent results with far fewer opportunities to make mistakes. It also reduces the number of opportunities for contamination.

Hey Phage 434,

you are actually right I will be doing cloning later on.

Thanks for the heads up I will have to order new after making sure they work
If I choose the following 5' 8 bases do you think they are fine as a start ..look below
5'(ATGCTATA)(c a t a t g) a t g g t g g a c t t g g c t a g g g t 3’ forward primer
5'(GTACTAAG)(c t c g a g) t t a g c t g g a c a a c a g c t t t t c a 3' reverse primer

Let me know what you think!
Thanks Again,
Yasamino

[Adrian K]

yasamino, on 18 August 2011 - 02:30 PM, said:

Adrian K, on 17 August 2011 - 09:39 PM, 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

[leelee]

yasamino, on 18 August 2011 - 02:44 PM, said:

almost a doctor, on 18 August 2011 - 02:33 AM, 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.

[gebirgsziege]

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?

[almost a doctor]

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!

[Fluffy]

Adrian K, on 18 August 2011 - 07:02 PM, said:

yasamino, on 18 August 2011 - 02:30 PM, said:

Adrian K, on 17 August 2011 - 09:39 PM, 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

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

[Fluffy]

leelee, on 18 August 2011 - 08:10 PM, said:

yasamino, on 18 August 2011 - 02:44 PM, said:

almost a doctor, on 18 August 2011 - 02:33 AM, 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