I am trying to genotype my transgenic mouse by following the protocol of a paper published on the original mouse data and cannot get a clear PCR gel. I want to genotype for the wildtype gene (Rad52) and the gene knockout which has a neo cassette in place of exon 3. The published paper lists the following protocol:
a primer specific for the neomycin gene (Neo-FB; 5′-CGCATCGCCTTCTATCGCCT-3′), a primer specific for MmRAD52 exon 3 (R52-45; 5′-AGCCAGTATACAGCGGATG-3′), and a primer complementary to intronic sequences downstream of exon 3 (R52-46; 5′-CAACTAGATACATGCCCACG-3′).
The PCR conditions were as follows: 1 min at 93°C, 1 min at 55°C, and 3 min at 72°C for 35 cycles. The product of the wild-type allele is 120 bp, and the targeted allele yields a 320-bp product.
I also tried a touchdown PCR method, starting at 71 degrees Celsius, with 17 30 second touchdowns until I reached 55 degrees Celsius and then continued with the aforementioned protocol.
For my master mix I use the following (per 1 rxn):
12.5 ul 2x Bullseye Taq plus master mix
5 ul 5M Betaine
.5 ul Primer 1
.5 ul Primer 2
4.5 ul H2O
2 ul DNA
I keep getting smeared lines and many times no band at all.
In my experience, many things can go wrong with PCR genotyping and you may have to try changing a variety of conditions before it works. A few suggestions:
1. Can you get good PCR using primers for other targets with your mouse DNA? If not, the quality of the DNA may be a problem. Also, sometimes simply using less DNA will make it work (perhaps use 2 ul of a 1:5 dilution).
2. You seem to be describing a three primer reaction in which you would get different bands for WT and KO, and both for the Het. However, you only list two primers in your recipe. Perhaps double check this. Sometimes separating a three primer reaction into two will solve the problem and sometimes a three primer reaction won't work as two separate ones.
3. Your PCR conditions seem unusual. Usually, a 3-5 minute step at 95 degrees is performed first, then the melting step is reduced to only 15 to 30 seconds on subsequent cycles ( I've also seen 94 degrees, but never 93 degrees). Also, 3 minutes at 72 degrees is unusual. The general rule is 1 minute per 1 KB of extension. So, since the largest band is 320 bp, you could cut this way down (I would try 30 seconds).
4. In my experience, the optimal annealing temperature can be different for different machines. If the other ideas don't help, try both higher and lower than 55. You should also look at the predicted Tm for each primer.
Good luck!
In my experience, many things can go wrong with PCR genotyping and you may have to try changing a variety of conditions before it works. A few suggestions:
1. Can you get good PCR using primers for other targets with your mouse DNA? If not, the quality of the DNA may be a problem. Also, sometimes simply using less DNA will make it work (perhaps use 2 ul of a 1:5 dilution).
2. You seem to be describing a three primer reaction in which you would get different bands for WT and KO, and both for the Het. However, you only list two primers in your recipe. Perhaps double check this. Sometimes separating a three primer reaction into two will solve the problem and sometimes a three primer reaction won't work as two separate ones.
3. Your PCR conditions seem unusual. Usually, a 3-5 minute step at 95 degrees is performed first, then the melting step is reduced to only 15 to 30 seconds on subsequent cycles ( I've also seen 94 degrees, but never 93 degrees). Also, 3 minutes at 72 degrees is unusual. The general rule is 1 minute per 1 KB of extension. So, since the largest band is 320 bp, you could cut this way down (I would try 30 seconds).
4. In my experience, the optimal annealing temperature can be different for different machines. If the other ideas don't help, try both higher and lower than 55. You should also look at the predicted Tm for each primer.
Good luck!
Have you checked whether the primer sequences are correct? It sounds stupid but sometimes published primers are from the reverse orientation.
Michaelas answer is excellent. Your PCR conditions do seem unusual for genotyping products that small. I routinely PCR genotype and would use 30 min to 1 min at 72c for products of that size. In terms of annealing temp try the Tm to Tm -2C considering your product is smeared
https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0
http://eu.idtdna.com/calc/analyzer
Hi Laurence, Thank you for your response. What do you mean by try the Tm to Tm -2C?
Sorry. I wrote that response on the train. I meant to say use tm -2C as your annealing temp.
No problem. What if my Tms for my primers are 60, 54 and 57 (forward, forward, reverse)
Hi Rachel. In a conventional 2 step or 3 step PCR with 2 opposing primers you would try and keep your primer Tms within 2C of one another. In a situation where your Tms span 54 to 60C the only way in my opinion of this possibly working would be touch down PCR. Your touch down cycles should start at Tm + 3 for your highest cycle and proceed in -0.5c to 1c intervals to Tm -2/3 c below your lowest Tm primer
Thus:
1. Start with an initial hot start denaturation cycle at 96C for 3 min
2. Then try 3 step PCR commencing with an annealing temp of 63C. That is 94c for 20 sec 63c for 20-30 sec and 72c for 30 seconds. Repeat this cycle but reduce the annealing temp by 0.5c down to 54c. That is 18 cycles. Then proceed with standard 3 step PCR for a further 15-20 cycles at 54C. Finally complete the PCR with a terminal 1 cycle at 72c for 15-30 min
Note: as pointed out by Michael your denaturation cycle although 93c is unusually long and this could cause some DNA breakdown and smearing especially if you have prepared your crude genomic DNA ( as I do) by crude lysis rather than full prot K digestion and column or trizol extraction.
If incidentally you continue to set degradation regardless of these cycle modifications and even after diluting down your gDNA try full purification to eliminate inhibitors. Also as mentioned take out your Betaine:
Betaine is designed to amplify more efficiently DNA by PCR that is GC biased. It could be that your region is indeed GC biased; hence the necessity for adding. However some labs add routinely regardless of GC content and on occasions I have known the addition cause problems especially in the context of crude genomic lysates rather than fully purified samples. That being so it might be salient to look at how the authors of the paper you quote prepared their DNA in addition to the PCR conditions. Good luck
Even when the PCR conditions are optimized, smearing occurs with old samples due to degradation. Hope you are working with fresh samples
Hi there. Andrew has provided an excellent addendum to my answer. To reiterate and append Andrews answer
1. The very best solution to your PCR as Andrew has said and I touched upon is to redesign your primers. You could try touch down with the existing primers; only touch down will work because of the disparity in melting temp but ideally especially if this genotyping is on going commitment it would be better to redesign new primers
2. As already mentioned make sure you are using 20ng to 100ng of your genomic DNA which normally equates to about 1-2ul of your final eluted genomic DNA ( per 50ul to 100ulof sample equate depending on how you have purified). Do not attempt PCR with more than 100ng as impurities may then exceed a certain threshold or molarity and actually start to inhibit your PCR especially if you are using a crude genomic prep which also contains contaminating salts ( see below)
3. Regarding purification preparation of crude genomic lysates by heating your tissue in 0.05M NaOH for 15 min followed by neutralisation with 1M tris pH 7.5 to 6.5 followed by an incubation for 4hr or more to allow the gDNA to leach from your tissue should suffice if you then take 1ul and utilise in a 20ul PCR reaction ( no more as the inevitable impurities in crude lysates could then actually start to inhibit PCR). This is how I routinely carry out transgenic PCR genotyping. In the context of your sub optimal primers however full purification with proteinase K plus either trizol or column might be necessary in conjunction with touch down to obtain good data
4. Regarding good primer design which would really provide a more permanent and fundamental solution to your problems as mentioned by Andrew and myself in my original answer I refer you to my good primer design document in this answer
5. In essence to design good primers as described in this document keep there Tms within 2C of one another and ideally at the same Tm:
6. Try and design primers with Tms of 60-65c and start with an initial PCR where your annealing temp is TM-2C. For well designed primers with matching TMs standard 2 or 3 step PCR will probably work. Indeed this is what I do when I routinely perform genotyping PCR on gDNA derived from transgenic mice
7. As described in my document and in fact in prior answers of mine on this forum pay particular attention to the last 5 bases of the primer: Try not to include more than 3 G/C residues in those last 5 bases. Furthermore do not end your primer with a double G/C residue. Rather an A or T. This will minimise primer dimer girmation
8. Screen for primer dimers using programs indicated in my document namely the IDT oligo analyser tool; also screen for intra primer self annealing; that is hair pin loops
9. For primers with a Tm of 60-65C you will probably find that your primers will be 18-21bp and 40-60% GC
10. Finally add betaine to your reaction if your target sequence has 50% or more GC where I know from personal experience this can make the difference between failure or success. If however the target sequence has a standard GC content or is not characterised by runs of GC bases omit betaine as this for standard targets in my experience ( and unlike GC rich targets) can inhibit PCR
The others have given good advice about concentrations of DNA (use less DNA as impurities can kill PCR). I typically use 10ng in 10ul PCR. Concentrations of primers matter too, especially in multiplex or three primer as they interact and interfere in solution. I sometimes decrease primer concentration to get reactions to work better. I typically use 0.1uM in 10ul PCR. Laurence indicated that betaine can kill PCR if GC content of primer is too low - I have seen this too - and wouldn't routinely add it unless you are sure of secondary structure from high GC content. Michael suggests separating PCR into singleplex until you have reactions working- this is good advice and we routinely do this in my lab for 3 primer and multiplex reactions. I have at times returned to singleplex when multiplex suddenly stops working too. Little things like one primer stock going bad can be hard to figure out in multiplex, and it can cause the whole reaction to smear. Smearing of PCR can be indicative of too low annealing temperature too, one suggestion is a temperature gradient increasing annealing from 55C.
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