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!
Have you checked whether the primer sequences are correct? It sounds stupid but sometimes published primers are from the reverse orientation.
Hi Laurence, Thank you for your response. What do you mean by try the Tm to Tm -2C?
No problem. What if my Tms for my primers are 60, 54 and 57 (forward, forward, reverse)
Even when the PCR conditions are optimized, smearing occurs with old samples due to degradation. Hope you are working with fresh samples
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