no PCR product from genomic DNA - (Oct/17/2006 )
Hi,
I am trying to amplify rather large fragments (5kb, 2.5kb) from genomic DNA and apperantly I do not get anything. I am using Phusion enzyme which is supposed to have high processivity and low error rate. Primers are OK. I would be very glad if somebody could give me a suggestion. By the way I use:50 ng DNA in a 50 ul reaction.
Thanks,
Could you please tell us your cycling condition and melting temperature of your primers. The sequence of your primers would be nice too... anotate said sequence
Can you do any short PCR reactions to show that your template is clean enough for PCR. Failing that, can you prepare 2 PCR reaction tubes, and add 1ul of genomic DNA, run the PCR reaction, run a gel and see if there is any inhibition of the reaction.
Is the template DNA still in good condition. Have you run a gel to confirm that the DNA has not been degraded.
Where was this genomic DNA obtained from. Please tell us what method was used to extract the DNA.
50ng is way too little DNA to use. I would use 200ng if not more (assuming the volume is kept small)
I have found the fusion enzyme is working under much different reaction conditions than other taq + proofreading mixes. You may need to play around with the conditions eg. MgCl2 Conc. to get it to work. I have also found that sometime adding in a small amount of regular taq can get the reaction to work and still take advantage of the proofreading ability of the fusion enyzme.
Can you do any short PCR reactions to show that your template is clean enough for PCR. Failing that, can you prepare 2 PCR reaction tubes, and add 1ul of genomic DNA, run the PCR reaction, run a gel and see if there is any inhibition of the reaction.
Is the template DNA still in good condition. Have you run a gel to confirm that the DNA has not been degraded.
Where was this genomic DNA obtained from. Please tell us what method was used to extract the DNA.
50ng is way too little DNA to use. I would use 200ng if not more (assuming the volume is kept small)
Hi,
Thanks for your answer. Regarding your questions:
One pair of primers I am using is as follows: (the colored part is the one that is hybridizing, previous nucleotides are the restriction sites).
1) ATC TGA TGC GGC CGC ATT AAA GGT GTG CAC TAC CAC
2) AGC ACT CGA GCA CTT CAT ACA AAG AGC TAC TG
With the above mentioned primers I am aiming to amplify 5 kb fragment and for this I use as annealing T: 57°C and the PCR program I am using is :
98°C - 1min
98°C- 30 sec x 25 times
57°C-30 sec x 25 times
72-2 min x 25 times
72°C-7min
98°C is the T that is suggested for this Phusion enzyme. I am not using more than 25 cycles not to mutate the product that I will get.
We have obtained this genomic DNA from our collaborator and they say that they isolated the DNA from the mouse tails.
About the DNA quality: I have limited amount of this genomic DNA
and therefore I am kind of being ungenerous to spend it (I have to optimize and then amplify 6 products from this DNA).I have not checked the DNA quality on a gel and I do not actually get what you mean by running short PCR with this DNA. CAn you please tell me what kind of primers then are used in this type of reaactions to see if there is inhibiton.
Thank you very much for the help,
Cheers
The Tm of your primers according to the IDT website is 53 degrees Forward primer and 50 degrees reverse. I think your annealing temp is too high for these primers to work. Remember it is only the part that hybridizes to the target that contributes to the Tm.
I'll just add similar to my previous reply, that the Phusion enzyme can be difficult sometimes based on my experiences. May want to keep that in mind. You can try the reaction with regular Taq as a positive control if you do not believe me.
according to oligocalculator, the tm for
primer1 = 57
primer2 = 58
so the annealing temperature used is actually low for phusion (uses tm +3). However a little low is okay for optimisation if the yields are low.
the melting time of 30sec is too long. Drop it to 15sec
increasing Mg ions could also improve the reaction.
nested PCR could also save the day.
But first things first, as DNA is limited, I have the following suggestions
The first thing you should do would be to confirm that the DNA you have is of good quality and not degraded. Run a small quantity of DNA in a narrow gel well. Run the gel well so as not have to repeat this sacrifice. (use clean gel box, clean gel, don't run the gel in a hurry-take your time, use clean loading buffer, running buffer... the works)
If the DNA is okay and not degraded. The next thing you need to know is if the DNA is contaminated. Set up a PCR reaction to test the quality of DNA. Do you have primers that can amplify a small fragment from this genomic DNA? If yes perform said reaction on a small sample of your genomic DNA.
If no, then set up three 20ul (total volume) PCR reactions (A, B & C) that amplifies any old piece of DNA you have. It doesn't matter what just some PCR reaction you know works. Add : -
0.5ul of your genomic DNA to tube A.
1ul of your genomic DNA to tube B.
no genomic DNA tube C
use Taq, 25 cycles
Run the reaction... if the genomic DNA preparation is clean, all three should give a signal of the same intensity.
If DNA is not clean.. ie the signals are not the same intensity, then clean up the DNA.
Also, 5kb is not that big. Other polymerase that can be used in this range are like KOD Hifi. It is relatively cheap. So you have a chioce if Phusion doesn't work for you. 4kb is about the limit of Taq... I am not sure if you can get a signal without optimisation. (Which would be a waste of time and limited DNA sample)
Finally a bit of personal preferance... I am not keen on the use of proof reading enzyme - taq mixes. They work for short DNA fragments 5kb... but for long fragments >10kb, the taq just introduces errors. Like yourself, I prefer using phusion + UNG mix, to get me over that hill safely.
Always assume the worst when somebody gives you DNA in a tube. It may not even be the right thing.
Look, we know that different TM calculators can give different results based upon the algoritim used. This has been discussed ad nauseum. However, I have found the Tm for the IDT website to be very reliable in my experiences. I am just pointing out that this could be an issue and would be a first step in trying to problem solve in my opinion.
98°C - 1min
98°C- 30 sec x 25 times
57°C-30 sec x 25 times
72-2 min x 25 times
72°C-7min
98°C is the T that is suggested for this Phusion enzyme. I am not using more than 25 cycles not to mutate the product that I will get.
I know this is a bit late, but is 2 min @ 72C a long enough extension time for a 5kb fragment? The protocols I have used (granted they are with Taq) suggest at least 1 min/kb. Could this be part of the problem?