Hi Guys:
I performed PCR to confirm if my gene knock-in is working fine. The templates are yeast genomic DNA. Primers' Tm are 45C and 60C, and I set annealing temperature at 55C for 30 second. The target PCR products should have 2400 bp. The genomic DNA has been 100-fold diluted before PCR, which is about 1-5 ng per 20ul reaction.
I have used new 1x TAE running buffer and another PCR product which the size is known (3300bp) as control.
But when I run a gel, all my PCR products just stick in wells and they almost didn't migrate. My control PCR product (3300bp) migrated at the right size. Marker looks normal.
Please give me any suggestion.
Do I need to dilute more for the template genomic DNA?
Thank you for the help. I really appreciate.
Jun-
All Answers (31)
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The PCR has never worked before correct? These are new designed primers, aren't they? Can you post also the primers, please? Do you have an image of the gel? -
Hi Ijad:
Thanks for reply. It worked once with very faint bands at the right size. But then it became to be not repeatable. One primer is now ordered (forward), the other primer (reverse) we have it and is stored for 3-4 years in freezer.
Forward primer: 5'-CTT GCT TTC CCA TCT CAG AG-3', with Tm: 53C
Reverse primer: 5'-GCC AAC CAA GTA TTT CGG AGT GCC-3' , with Tm: 60.5C
I will post the image here by tomorrow. The image is like Bright bands are in wells.
Thanks for any help.
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My guess is that the primer are not binding. The GC Content of both primers is fine and the 3' ends are also ok. Maybe others here have another idea. Even if it worked once with a weak signal, this also means that the binding is not working 100%.
Maybe anyone else from here has another idea?
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Hi,
I don't know about yeast, but when I was doing PCR in porcine genomic DNA I usually used 100 ng of genomic DNA as template. Another thing that can help is adding DMSO to the reaction.
Good luck -
Ignacio,
DMSO makes sense ;).
-Ijad -
Thank you guys. Someone said it may be the preparation of genomic DNA. I used 100ng of genomic DNA of this preparation and used another pair of primers which gives me 800bp PCR products; it works perfect. I can see bands at 800bp. But it just did not work when using primers for 2400bp PCR products. I am confused.
However, I will try DMSO. Thanks for suggestion, Ignacio. -
Hi
maybe you can increase the time pcr, 1 minute -
JH,
how long is the elongation phase of your PCR program?
-Ijad -
95C, 30sec, 55C 30sec, and 68C for 3min. for 30 cycle.
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there is maybe more than just one problem with your PCR...the fact that you have had already a product of right size is a marker that it could work, but the conditions need to be optimized.
in total reaction volume never go under 10ng of genomic DNA, 20ng is better, 100ng should work perfectly. if it doesn´t, try additionally an annealing T of 58° or even 60° (55° is too low for your reverse primer). the product is very long... which polymerase do you use? try one that is specifically for long products. many companies serve also special buffers for these polymerases. that always works.
another tip: if you want to store primers for longer dilute primers not in water! they can get broken, use buffers. maybe you also need to order a new reverse primer... -
According to me i guess there are following possibilities
1) the gell loading buffer which you used for loading the sample is not proper.
if you have used he same gell loading buffer for the markers and your control then this possibilty is ruled out
2) PCR is not being carried properly.
this mght be because the designed primer pairs are not proper. Tm difference should not be more than 5 degree celcius. In your case the Tm of your primer is 45 degree and 60 degree but you have set the anneling temperature at 55 degree. In such case only the primer with the Tm 60 will anneal and not the primer of 45 degree. this will result in amplification of single strand.
But as in your case you are getting a bright band, this could be due to genomic DNA; indication negetive PCR amplification.
So i will suggest that check the gell loading buffer and redesign your primers with Tm difference not more 5 degree celcius.
If you want to check if your PCR product is single strand amplification then perform PCR in two sets. in one set add both the primer and in the another set add only the primer with Tm 60 degree. keep the condition same as you mentioned above. keep annealing temperature 55 degree for both the set. observe the result.
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Hi Jun,
could you show me the amplicon sequence? What is the time of elongation step?
Increse the tempature of denaturation step (98° for 20''), alternatively, use a touch down PCR. (65° to 54°)
Good luck. -
Hi Jun,
you need to first optimize your annealing temperature by doing a gradient pcr (50 to 65C), the absence of product can indicate the need for a lower annealing temperature.and increase the annealing time.
As a general rule, extension times of one minute per kb should be used. A final extension of 5 minutes at 68°C is recommended.
good luck. -
hey JH
the extension time is not an issue..and i wouldn't suggest using DMSO..you're just complicating your experiment...why not try decreasing the annealing temperature to 50 as one of your primers has a Tm of 45?
if that does not work i suggest you redesign one of your primers. add a few bases to the primer with Tm 45 or remove a few bases for the primer with Tm 60 -
Hi Jun, still in the lab?
Linda and Siddarth made a good suggestion. Jasmin´s comment was also important: don´t use water when storing primers for a longer time. As only your forward primer was newly synthesized and the reverse one is pretty old I would suggest to order a new reverse primer. Possibly you can choose primers with similar Tm´s on that occasion? Would be much better. What do you see if you apply a sample of your PCR reaction on a gel before doing the amplification? What happens if you try one or two different polymerases (like Phusion). We need your input now. -
Why not try to optimize or adjust the conditions of your reaction. -
A primer with a Tm of 43 degrees will not bind DNA at an annealing temp of 55 degrees. The annealing temp should be a few degrees lower than the Tm of the primer. Also, the template DNA could be increased to 50-100ng. -
Also, ideally a primer pair should have Tm's within at least 5 degrees of each other if possible.
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Thank you everyone for all the comments. They are very useful. Thanks for Jasmin,Siddharth, Michael, and Jorg's comments. The primers are stored in water, so as I think that is why it doesn't work properly. The problem must because the designs of this pair of primers. I will redesign the primers or just increase annealing Tm to 60 and increase annealing time.
Jasmin, the polymerase we use in our lab is Klen Taq, which is the only one we usually use for PCR amplification in this lab. It has its reaction buffer. the product length is not a main issue. It never worked when I use this pair of primers to amplify and to check the KnockIn gene (2400 bp PCR product) or the KnockOut gene (will have 800bp PCR product). However, these primers work properly when they were pair with other primer individually.
Manojkumar, the DNA loading dye is okay. Other PCR product work normal when using the loading dye. Tm is absolutely the main problem in my case.
It probably is better to redesign primers than keep trying all different PCR conditions.
Thank you guys for these great suggestions.
JH -
add DMSO and NH4 positive ions will open your negative loaded DNA and let your primers access the GC rich "DNA spaghetti"
or simply buy MyTAQ that has it all in the buffer optimized better than any other TAQ supplier... -
Hi everyone, I attached a gel here and take a look if you are interested in. Thanks, JH
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I had the same problem several times. Looking at the picture I can say that is a lot of DNA. Defnately amplified! I would guess PCR is fine. Try diluting the sample and run the gel that sholud give you an idea where to fix it.
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Are you loading a PCR product? IF so, you need to dilute it more or add less prouct totthe wells. If you're loading genomic DNA load WAY less! If the PCR did not amplify you're running genomic DNA. TOO much DNA will prevent PCR amplification. Try to use NanoDrop or plate reader to quantitate your genomic DNA and PCR product.
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From the image all i can say is that the DNA load is too much. you need to dilute the sample before loading and also dillute the templet DNA appropriately for the PCR.
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Hi!
As per my experience by seeing your gel image I say that it is not PCR product stick to wells, it is some junk with high molecular weight. Literally PCR did not work, see the middle four control samples, how they worked.
As you mentioned generally primers are dissolved in water and will be stored in freezer, other wise they degrade after some time. Use the correct set of primers and you get amplification. -
Jetty, thanks for your advice. Primers condition may cause the problem. That's a good point.
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How many cycles of PCR are you using? 30 or more cycles may be too much and be generating high molecular weight concatamers or other complex DNA structures. Try setting up multiple reactions, pausing the cycler and taking out after 15, 20, 25 cycles for gel analysis. If you are adding ~5 ng of yeast DNA template, it should only take 20-25 cycles to get a strong band with an efficient PCR primer pair. -
JH have you quantified your template DNA? from the image it looks like you are adding way too much template..why don't you try a 1:10 serial dilution (1:10,100,1000,10000 etc) series and check?
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I would say this is some sort of High Molecular Weight DNA that generally can get stuck in the well. This is not a PCR fragment. -
If DNA amount is too much, you should see a smear, but the band should still run at proper size. Please try to use less template DNA and optimize the annealing temparature as well as cycle number. Please also try to add DMSO (1ul or so per 25 ul reaction mixture), which sometimes clears the band by making the primers bind properly. Also, if you are keeping the PCR product in cold, try warming it us around 60 degrees, right before running the gel.
Popular Answers
1) the gell loading buffer which you used for loading the sample is not proper.
if you have used he same gell loading buffer for the markers and your control then this possibilty is ruled out
2) PCR is not being carried properly.
this mght be because the designed primer pairs are not proper. Tm difference should not be more than 5 degree celcius. In your case the Tm of your primer is 45 degree and 60 degree but you have set the anneling temperature at 55 degree. In such case only the primer with the Tm 60 will anneal and not the primer of 45 degree. this will result in amplification of single strand.
But as in your case you are getting a bright band, this could be due to genomic DNA; indication negetive PCR amplification.
So i will suggest that check the gell loading buffer and redesign your primers with Tm difference not more 5 degree celcius.
If you want to check if your PCR product is single strand amplification then perform PCR in two sets. in one set add both the primer and in the another set add only the primer with Tm 60 degree. keep the condition same as you mentioned above. keep annealing temperature 55 degree for both the set. observe the result.
Maybe anyone else from here has another idea?