Hello fellow scientists,
I could really use some advice! I'm using a new(ish) Bio-Rad T100 thermocycler to run a mutagenesis PCR using NEB's Q5 Site Directed Mutagenesis kit (E0554S). My PCR results are highly variable! Sometimes one sample works and another one doesn't, sometimes the other sample works and the first one doesn't, sometimes the positive control fails, sometimes all samples fail, and sometimes all samples work. Sometimes the samples are weaker yield or robust yield. Every time, I swear I'm doing the exact same thing, in the exact same way, using all fresh and good reagents. I've done the experiment 10-15 times now. :(
Has anyone else had this problem of variable PCR results despite the exact same conditions, and have you found a way to get consistently robust PCR results?
I've also tweaked some of the experimental setup variables, hoping that it would provide consistently good results. Here's what I've tried: First, I've spoken extensively with NEB and Bio-Rad. I'm using 3 different primer sets on 2 different templates. I've checked every single reagent for the correct concentration and purity, and used fresh stuff (fresh kits, primers, template, even the water!). I've done replicates and master mixes. I've loaded the tubes into different wells of the thermocycler, I've done gradient and static annealing temperature runs. I've used empty insulating tubes in the gradient run. I've read the logs of the thermocycler, and they look good. I've played around with ramp rates of the annealing temperature. Before you ask about optimizing the annealing temperatures, allow me to mention again that sometimes the samples work great, and sometimes they don't work at all, and sometimes they work weakly, even with the exact same experimental setup.
Is mutagenesis PCR just inherently stochastic??? That would be maddening, but I would accept it and move on.
All advice is very welcome!!
one common reason for this is that the annealing temperature of one primer is just on the edge of working/not working so even small temperature variations in the pcr can work or fail.The thickness of the pcr tubes may vary slightly or even the total volume changes with pipetting errors may mean some samples reach temperature slightly quicker than others. The salt concentration in the pcr mix especially magnesium may have an effect on the annealing temperature. One other possibility is that one primer is degrading especially if dissolved in water and not TE. I would try newly produced primers if yours are old and inherited and set the annealing temperature 2c lower and use a more conservative cycle eg slightly longer at 94 and slightly longer at annealing and possibly slightly more mahnesium since Mg shortage results in poor enzyme activity. Use the inside wells of the pcr machine not the edges. If your failures lie together in the tube strip then there may be a problem with the heating peltier junctions in the block. They are usually in rows of 2 or 4 and a block can fail in one part but not all across the block but this option is probably not the cause but it does happen.
The difference between a successful pcr and an apparent fail is only a couple of percent efficiency in each cycle so parameters do not have to change much to be disastrous eg at 98% efficiency not 100% you only get 50% of expected yield after 30 cycles of pcr
It is also important when using pcr master mixes ( or any reagent really) to thaw then mix well otherwise at half melted the ice floats and all the salts are at the bottom of the tube then pipetting can either result in too much or too little reagent depending on where the end of the pipette tip is in the reagent solution
Dear Amber,
do you have the oppurtunity to make a test in another Cycler ?
In my experience the Biorad cyclers are the worst I have ever used.
I had exactly the same reaction in another cycler from peqlab and got much better results.
kind regards
Stephan
If you are not getting consistent results with your positive control, you need to troubleshoot your protocol without samples. This can be done with the suggestions from Paul Rutland & Stephan Schie
Your positive control is good positive control as far it is always amplified. Fix the protocol and then move to experiment. You can save your sample at least by this strategy.
Hi Amber Joy Vincelli if the positive control fails that is a sign either some reagent in the kit is not working properly or the PCR machine needs some care. I have lots of experience with site-directed mutagenesis with Agilent’s QuikChange Lightning kit (https://www.agilent.com/en/product/mutagenesis-cloning/mutagenesis-kits/site-directed-mutagenesis-kits/quikchange-lightning-233118). It works great in my personal experience but, once I had a horrible nightmare that is worth sharing here. The same kind of mutation that I was doing in different constructs suddenly stopped working with a new kit. The problem was due to a faulty kit. Later on, when I used another new kit, the problem was solved. As a rule of thumb, surely, introducing mutations in a construct needs standardization that can be positive or negative nevertheless, the positive control should always work. The experience that you have with NEB customer service makes me annoyed, they should have suggested you changing the kit as it was suggested to me in this situation.
Thank you Paul Rutland for taking the time to provide these thorough recommendations! I agree about the annealing temperatures, though I forgot to mention that I've used 4 different sets of primers (including the positive control primers supplied with the kit), and they all produce variable results. I am using thin-walled tubes specifically made for PCR. The salt concentration seems to be adequate, sometimes!! I am careful to thaw the reagents completely and mix thoroughly before pipetting. The primers are less than a year old with minimal freeze/thaw cycles (and the kit primers are very new). I hadn't thought about tweaking the cycle temps/times to be more conservative, I might try this! I do use the inside wells of the PCR machine (and a bracket around the outside so that the lid sits flat on top of the sample tubes), excellent point. I've also spent an hour or so using a small thermometer to measure the temperatures of water-filled PCR tubes while the thermocycler is on, but I couldn't identify any issues (or my thermometer was not sensitive enough). Great point about the small decreases in efficiency leading to large impacts in the yield! It must be something small...
If I ever figure out what's going on, I'll post back! Thank you again for taking the time.
Stephan Schie Great suggestion to try another thermocycler! Somebody else suggested this to me too. It's not so difficult to beg a neighboring lab to use their thermocycler for one run, I'm sure they wouldn't mind. :)
If/when I figure it out, I'll post back.
Sayanta Bera Actually, NEB was super helpful and sent me about $200 of new reagents for free! They are the only reason that I was able to continue troubleshooting the PCR. :) Sadly, I'm still getting highly variable results with the new kits.
Thank you for recommending Agilent’s QuikChange Lightning kit, I might try that instead if I can't get NEB's kit to work! I'll post back with the outcome.
Abhijeet Singh I agree, you are wise! My problem is that twice now I've done the PCR with only the kit's positive control (and negative control), and it worked perfectly. Then I repeat the exact same PCR with my samples included, and the positive control fails... But sometimes my experimental samples work... But sometimes nothing works... It's hit-or-miss, without a reason that I can see. I even load the PCR tubes in the exact same wells of the thermocycler, and it's variable.
If I ever figure it out, I'll post back! Thanks for taking the time to help me out. :)
Amber Joy Vincelli
Apart from your problem, I appreciate the way you communicate and provide feedback to the suggestions. Here on researchgate, mostly we see questions from people who were just asking for the sake of asking. They do not actually understand the problem, not formulate the question well and do not comeback with the best solutions.
This question of yours, and the related discussion will be very helpful for anyone having same/similar problem and found this discussion.
I wish you success soon!
Amber Joy Vincelli that's so nice from NEB. So, we can rule out the option of a faulty kit. With my Agilent experience, I can say that the plasmid and primer concentrations were critical to getting a reproducible result. The kit recommended using HPLC purified primers for higher efficiency and indeed it worked better than desalt purified primers. Depending upon the size of the plasmid, the amount required for doing the mutagenesis also varies (in my case for 10 kb plasmid, I have to use 30 ng of plasmid). The PCR product was treated with Dpn1 and then 10ul was loaded in the gel. If I saw a band (faint/bright), I proceeded with transformation.
Hi all, so just a quick update, in case others are doing similar troubleshooting...
I tried a neighboring lab's Bio-Rad T100 thermocycler (they have the exact same model as ours, lucky me!), and there was no improvement in the runs with their machine. The run logs look similar too (same ramp times, etc.). So, I don't think the problem is related to our thermocycler. I'll keep chipping away at this problem, and post back if I have more news!
As a side note, Synbio Technologies in NJ does mutagenesis for about $65 per sample, plus shipping (it's actually gene synthesis and cloning, but it's marketed as mutagenesis). I've used them for one mutation so far, and they were great! I haven't tested their sample yet for protein expression, but I'm confident that Synbio did the mutagenesis correctly and that they sent me quality DNA. product. I probably should have outsourced this a long time ago...
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