Another primer dimers problem - (Aug/17/2011 )
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
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
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.
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....
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.
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!
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!!!
gebirgsziege on Thu Aug 18 11:33:50 2011 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 phage434 on Thu Aug 18 12:42:47 2011 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
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.invitrogen.com/content/sfs/manuals/taqrecomb_pps.pdf
http://tools.invitrogen.com/content/sfs/manuals/taqnative_pps.pdf
http://tools.invitrogen.com/content/sfs/manuals/accuprimetaq_man.pdf
http://tools.invitrogen.com/content/sfs/manuals/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