i don't need Taq polymerase. Just need normal DNA polymerase. So the main idea is preparation polymerase directly from Lactobacillus without using cloned vector. My idea is electrophoresis using agarose gel and calculating the isoelectric point of Polymerase based on protein sequence to know where they will migrate after electrophoresis. Is it possible for this idea ?

  • $\begingroup$ I think it would be easier to use heterologuous expression of a tagged protein, e.g., a His-tag. You could then easily extract that protein from the lysates using a Ni-column. There are many more tags available, but the His-tag is the most common one. $\endgroup$
    – Eekhoorn
    Jul 12 '13 at 11:30
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    $\begingroup$ I guess this is still related to your DIY project, you should specify it, otherwise people will assume you are in a standard lab with full access to state of the art equipment. 'Normal' DNA polymerase is not thermostable. It will be inactivated at the first cycle of PCR, that's why most polymerase we currently use comes from organisms that live in very hot environments. $\endgroup$ Jul 12 '13 at 11:34
  • $\begingroup$ @GianpaoloR I'm running another experiment so taq polymerase for PCR is not necessary now. In this experiment i need to using normal DNA polymerase because normal DNA polymerase can synthesis in room temperature but taq polymerase not. No matter if other method could help me to learning more about biology even it's impossible for me. $\endgroup$ Jul 12 '13 at 13:26

This is a very ambitious project for a DIY-er. To explain why, I'm going to use the example of DNA polymerase I from E. coli.

DNA polymerase I is the most abundant DNA polymerase in the E. coli cell, but nevertheless is still only present at around 300 copies per cell

Ishihama Y et al. (2008) Protein abundance profiling of the Escherichia coli cytosol. BMC Genomics. 9:102.

This was the first DNA polymerase that was ever purified, in the laboratory of Arthur Kornberg

(Enzymatic Synthesis of Deoxyribonucleic Acid : I. PREPARATION OF SUBSTRATES AND PARTIAL PURIFICATION OF AN ENZYME FROM ESCHERICHIA COLI Lehman, MJ et al. (1958) J. Biol. Chem. 33:163-170.).

In this paper a method of purification is described that starts with 60 litres of culture. There is also this statement:

"One kilo of E. coli yields less than 10 mg of the purified enzyme."

I'm guessing that you have no experience of protein purification, so I'll just tell you that this is very discouraging: these days, with overexpression, people expect to get mg quantities of their protein from just a few grams of bacterial cells.

This is a clear illustration of why it is pretty much unthinkable for you to set out to purify an enzyme like this using small-scale methods, and explains why techniques of overexpression and the use of affinity tags have revolutionised protein purification.

And it gets worse: even if you did manage to purify some DNA polymerase I from a bacterial source you would find that it is actually not very good at making DNA. This is because as well as extending primers in a 5'>3' direction, it is is very good at degrading DNA in the same direction. In other words as the enzyme moves along a template molecule, copying it, it will degrade any existing DNA that it meets "ahead" of its direction of travel. This is why the most commonly-used form of this enzyme in research is the "Klenow fragment", a fragment of the polymerase that lacks the 5'>3' exonuclease activity. This fragment was originally made by treating DNA polymerase I with the protease subtilisin, but it is now expressed from an engineered polA gene. Klenow fragment polymerase was used in the original PCR experiments (it had to be added afresh at every cycle).

I don't know how much money you have to spend on your projects, but in fact these enzymes can now be bought relatively cheaply. I don't think that you can expect to make them for yourself, without this becoming the actual project.

Finally, if you can get hold of a strain of E. coli with an expression plasmid for Taq polymerase then you really could make your own very easily - I've done this myself (but I no longer have the strain) and the heat stability of the enzyme makes the purification trivial. It would be much easier to use your own Taq with a heated water bath than to try to make DNA polymerase I. But obviously it depends upon what you are trying to do: Taq polymerase may be unsuitable for other reasons.

  • $\begingroup$ Incidentally - I perhaps haven't made it clear that although you don't need mg of enzyme for typical molecular biological applications, it is impractical to purify very small amounts of a protein. $\endgroup$
    – Alan Boyd
    Jul 12 '13 at 20:55
  • $\begingroup$ Thank Alan Boyd so much, you always give me a quality answer for my question and give me a direction what i need to do next. I'm planning to create a DNA sequencer, the main idea for my project is heat the tube to 95 oC -> 60 oC for primer can bind. And then cool down to 37 oC for normal polymerase can work. I need temperature room because i think one of my sensor chip can not work in high temperature. I never work with plasmid before and it's seem that i need to get used to it. $\endgroup$ Jul 13 '13 at 2:35
  • $\begingroup$ I'm an IT engineer and all of my worked money is being spent for research biology. A lot of money was spent and i want to save money as possible for my future research. Enzymes are very expensive for me as the price i see in this table at enzymatics.com/products.htm . However it cannot make me downhearted. $\endgroup$ Jul 13 '13 at 2:57
  • $\begingroup$ @ĐứcUltraSoft: if you want to do some research then the best option would be to work as an intern in some lab where you may also implement your ideas. Biological research especially molecular biology is a costly affair and you may end up losing more money. I think that you can write to George Whitesides who is in Harvard. His lab is into bio-entrepreneurship. $\endgroup$
    Jul 13 '13 at 5:09
  • $\begingroup$ @AlanBoyd do you how many molecule of taq polymerase is needed for a PCR reaction ? $\endgroup$ Jul 18 '13 at 11:58

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