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0mg_Its_Sabby 0mg_Its_Sabby
wrote...
9 years ago
Trying to understand why DNA purification was important to a lab experiment last week where I purified the DNA then ran a gel, but what I'm not grasping is why it was important to do. The bacterium used was E. coli .
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wrote...
Staff Member
Educator
9 years ago
That gets rid of all the unincorporated nucleotides and primers, and allows you to put it in a buffer that is compatible with the next enzyme. If your PCR reaction isn't 100% specific, and you have more than one product produced, you can gel separate the different sized fragments by agarose gel electrophoresis, cut out the band that you want, and purify it from the agarose.
Mastering in Nutritional Biology
Tralalalala Slight Smile
wrote...
9 years ago
I just do purification of DNA before ligation or transfection.

(1) for ligation, the 'salt' (Nacl,Mg2+,Tris-HCl etc) can influence the ligation. The buffer for ligation is different from the buffer for enzyme digestion or PCR, so that we need to do the purification before ligation to get the DNA in elution buffer or di water.

(2) transfection, the endotoxin is not good for cells so that we need to purify the DNA to get rid of endotoxin (we normally get it from bacteria when we amplify pasmid in bacteria)
zoeksyrianos Author
wrote...
9 years ago
The DNA is purified to make sure you isolate one cell's DNA only. The DNA would then be "amplified" so that it can be detected.


DNA is very seldom isolated from a single cell. Once isolated it cannot be used to tell the persons hair or eye color - you would need to have very specific probes for a lot of genes, there are many other problems here in using it to identify anyone. IF you are looking at a crime the DNA could be used if you have another persons DNA to compare it to. If you are given DNA and no suspect - you're out of luck. These comparison are done by looking at digestion of DNA samples with a variety of highly specific endonucleases.

Isolation of pure DNA is normally done in a research lab so that it can be studied without interfering substances. such as RNA or protein that might inhibit or block enzymatic digestion of the DNA or amplification of the DNA by PCR.
wrote...
9 years ago
Many times in forensics, microbiology and genetics the analysis of DNA is needed. However, more times than not the amount of pure DNA available is minimal. There for scientist routinely will take a sample containing the original DNA, use it as a template and run it through a procedure called PCR (polymerase chain reaction). This is basically like using a copy machine. At the end of this process you have multiple copies of the original template.

Now think about it, if your original template is contaminated the copies will also be incorrect. Which can lead to improper interpretations and in the case of forensics can free a true criminal or worse land an innocent person in jail.

For this reason DNA is purified. Purified in the sense that samples may contain eroneous fragments of other DNA and, or other nucleic debris which can constrew peak readings. Think of it this way, when cooking a sifter or strainer is used to collect all of the same items based on factors such as size. In the same way purifying a sample collects all of the same DNA.
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