I am at my wits end with this PCR so I am hoping someone in the world might have some ideas. I am consistently getting inconsistent results.
I am following the PCR protocols adapted from " Joung, J., Konermann, S., Gootenberg, J. et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc 12, 828–863 (2017). https://doi.org/10.1038/nprot.2017.016"
I need to use these 2 PCRs to amplify and subsequently analyze DNA from a CRISPR screen. I am using the above PCR protocol and primers that have already been optimized for this purpose and have worked successfully previously for our lab. Importantly, I am using herculase which is a high fidelity (expensive) polymerase, which I also heard can be unstable.
When I first tried to amplify this set of DNA as smaller scale PCR test for for PCR 1, I saw no bands except for the genomic DNA extracted, so no amplification.
I tried this again, this time comparing the amplification of a DNA sample that had worked pretty well before (enough DNA extracted from gel to mantain coverage)--> still no bands except genomic DNA.
(I had tried this before but had not maintained enough DNA after PCR2 for enough coverage of the screen and had to repeat again with some new DNA I extracted.)
I tried once again but using a different herculase (we have 4 separate vials) for each reaction to see if any had lost their activity. The DNA sample that used to work and a DNA sample from a colleague that he claimed amplified.
Now, I am either receiving very bright bands or no bands at all (at the expected ~300bp size) but there doesn't seem to be a pattern as to why. The herculase that didn't work before for my one DNA sample, now works but not for my colleague's sample. Some of the herculases work some of the time but not others.
I repeated again, this time I had someone else in the lab (AG) try it as well alongside me (EP), using two different DNA samples that worked well enough before (enough DNA extracted from gel to maintain coverage).
We both observed the same inconsistent results. This time instead of observing all or nothing I am seeing some wells with faint bands, some with bright bands and some with no bands.
I am mixing the components, mastermix and PCR tubes well before amplification and before loading onto an agarose gel. I see the same results when loading some of the sample into multiple gels so no problem with that.
I don't understand why this can be occurring.
If some of a reagent went bad it should be well mixed so that all samples get about the same amount of it, not leading to inconsistent results.
I have also previously checked (with 192 PCR reactions for a PCR reaction that always works with hotstart taq pol) that the PCR machine works well and there are no problems with pipeting.
If I cannot fix the problem and ensure I am always observing good, consistent amplification, I will be at high risk of not getting high enough coverage for my screen.
Let me know if you have any ideas or you would need more details.
Anyone have experience with Herculase and notice this before?
Thanks!
Eli
Dear Eli Perr
Hope you are doing well. It seems something is there behind the door. But I'm not getting it fully. What if you run the same sample repeatedly. Hope you can fix the problem soon.
Thanks and regards
Marzan
Dear Eli Perr
I was just wondering about the fluctuation and inconsistent result you’ve faced. Hope you can find the way out.
Hi I have done plenty of PCR previously but not with Herculase. But here are my thoughts.
So :You prepare a master mix (s) so that all samples have the same components. you test different preps of sample, both you and others and you both get inconsistent results. If Herculase is unstable this may be a problem: but I'm not sure why. However, have you checked the PCR machine in terms of temperature control across the whole unit. I would repeat PCR with the same sample that previously worked (or you are most confident about) and distribute tubes across the block (perhaps 6-8 reactions) or use another basic PCR using Taq to save money. Make a note of positions in block. check results. Repeat again...do you get the same results? If you have got the same results using another PCR machine then it is unlikely to be the temperature control in the block.
hope it resolves for you.
I might be wrong but I noticed in your gel image the primer dimer band (usually seen in the bottom of the lanes is very faint and where ever you don't have a positive amplification band there the primer dimer band is fainter. I would recommend heating your primer stocks at 50-65 degrees celsius, followed by thorough mixing and spin down before using it for your PCR reactions. That way you can ensure that the primers are uniformly distributed in the primer mix and heating ensures proper dissolution of individual primer molecules (minimizing dimerization).
I usually aliquot my working stock of primers in 50-100ul volumes, in multiples of 4-5. And use only 1 vial at a time, to avoid the risk of contamination and loss of primers due to repeated heating. This was the only thing I could think of to suggest to you. Hope your problem will be resolved soon. All the best.
Nidhi .O The PCR seems to be working for now, though I am not sure what was the fix. I grabbed a new set of dntps and rediluted my primers. I always thought I mixed very thoroughly (vortex and spin down) but I am now vortexing for longer just to be sure. I also add the herculase last, only removing it from the freezer at the last moment (I previously kept it on ice).
I think the heating of the primer stocks, that you suggested, may also be a good idea.
Expertise did not grow in a time. So, do it repeatedly by following the methods from the various good articles. It also needs a good extracted DNA/RNA which are free from alcohol, debris, or others.
I am sharing the same thought of Nidhi.O and spinning of each master mix component is well-considered during master mix preparation.
Eli Perr I'm curious to know the quantity of DNA you are using in PCR reaction because sometimes a high concentration of DNA may cause inhibition to PCR reactions. If this is the case then dilute the DNA. I hope it will work.
Regards
Hi Eli Perr . Based on you June 18 reply, it sounds like you had primer degradation and/or dNTP degradation. Like Nidhi .O suggested, making small aliquots of your primers and limiting the number of freeze thaws. After a 15 or so freeze-thaws, we threw away whatever was left (and reduced future aliquot sizes if needed). We also would throw out dNPTs or primers if we saw any precipitate at the bottom that did not immediately dissolve. Thoroughly vortexing and warming up the primers and dNTPs is key. Vortex the master mix before adding the polymerase. Keep the polyermase (no matter what kind) in the freezer till the absolute last second is wise. Lastly, many delicate polymerases cannot stand to be vortexed, so after adding polymerase to the master mix pipette the master mix up and down gently to mix. I also think that Sandeep Kumar has a good point. I had a pain in the rear PCR where we finally had to nanodrop (spec) every DNA sample before the PCR and add similar amounts of template to each reaction based on the nanodrop results. Hope one of the above helps. Good luck!
- Badges
- Science method