Good thing takes time

These days I tried so many times to get my POT1 PCR product to get reamplified by the same PCR condition used to generate itself from circular POT1 plasmid. (Fig 1. you can see lane 3 and 4 only have primer but not 1.9kb band!)

The reason why I want to do this is I cannot get the POT1 PCR digested with BamH1 ligated to the pcDNA5/HA/MCS/eGFP clone (also digested with BamH1). After awhile, I star to think about my POT1 PCR product has some problems. Therefore, I try to reamplify the POT1 PCR product.

I tried different renature time (58 to 68), different extention time (1min to 5min), dilution of my POT1 PCR product (1/10 fold).... just cannot get it work. THAT IS REALLY BOTHERING ME.....

To solve the problem, I finally sit down and think about it. Since the problem is after I got my PCR product from circular plasmid.
1. whether the PCR product has some inhibitor in it due to the PCR clean kit?
2. whether linear duplex DNA is very differnt that circular DNA like Tm to perform PCR?

As shown in Fig.2: I digested the POT1 vector and plasmid with Pst1 (this site is in the pcDNA5/FAT plasmid not in the POT1 gene), and digested the POT1 PCR product with EcoR1 (this site is in the POT1 gene). The result shows clearly that the PCR product can be digested and the two bands add up similar to 1.9, which suggests the PCR product is really a POT1 gene and works fine. That eliminate my concern 1.

Later I performed PCR using these sample compared with original DNA samples. In addition, I tried again the previous POT1 PCR product, just want to put it as a negative control. As shown in Fig 3, surprisingly, the Pst1 digested POT1 can generate a 1.9 kb band, so the linear DNA is indeed a PCR substrate, no doubt about it! More striking is the POT1 PCR sample can also generate a 1.9 kb band this time!! (lane 5). Also, the PCR product from lane 3 & 4 can go to a second round PCR just as good as its parental ones.

So, suddently, everything turns out to be as what one would predict ! So , what is the problem for this month I 've been througn??????

One thing is differnt in this time's experiment: "The denature time increases to 3 min". After reasoning, I think this one can explain everything. The linear duplex DNA did have higher Tm compared to circular one right?.. the topology problem weight in. Although 94 degree is definitely high enough for all kind of dupelx lenght. but one thing I did not realize, it takes times to reach equilibrium. I think 2 min is not enough to denature a long duplex DNA, the Tm of primer is much lower than it (60 vs. 85). So when the temperature cool down from 94 to 62, the long duplex can replace the primer just before polymerase can access the end. Therefore, by taking longer time, the long duplex did seperate, and the time it take to aneal is more than the time Polymerase can access the primer.

啟示:
Even though , I haven't got my clone yet. But this result really stimulates me.

1. When I encounter a problem, don't try to repeat it again and again, ... THINK ABOUT IT FIRST...... This is an important attitude, a habit, a philosophy. Narrow down the question to specific points, designing practicle way to answer these questions, therefore I can test the hypothesis and increase my knowledge and experience.

2. Always keep thermodynamics and kinetics in mind, think about question by this angle. What should it be in this condition? where should it go, becomes? What does it indicate?