Gel Electrophoresis: Mistakes, Conditions, Samples - Help Needed

[MusicMonkey]

1. A student loads his samples on an agarose gel, but has hooked up the electrodes incorrectly (ie. mixed up the positive and negative wires). What would happen? If the student discovered his mistake after 5 minutes, and correctly hooked up the wires and let the experiment continue, what would be likely to occur?

2. Under what conditions would you use an agarose gel that is 2% rather than 0.8%?

3. What would we have put the EcoR1, HindIII and BamHI samples in 60C?

Please help. I did try and have been trying to answer a whole bunch of questions and these are the only ones left. Thanks.

 

[iansmith]

1. A student loads his samples on an agarose gel, but has hooked up the electrodes incorrectly (ie. mixed up the positive and negative wires). What would happen? If the student discovered his mistake after 5 minutes, and correctly hooked up the wires and let the experiment continue, what would be likely to occur?

What is the charge of DNA? In what direction would the DNA moved in your gel under normal conditions?

For the last part, it will depend how much your DNA moved. The larger the power the more the DNA moved. So you have to think that the DNA fragments will not start at the same point so think about the size and the matrix effect.

2. Under what conditions would you use an agarose gel that is 2% rather than 0.8%?

It has to do with size of DNA fragment and matrix. 0.8% is usually the gel use the separate a wide diversity of bands. A 2% is to separate smaller fragments and will give a better resolution for small fragments. Look at the agarose gel picture on that page http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html

3. What would we have put the EcoR1, HindIII and BamHI samples in 60C?

Increasing the temperature during restriction enzyme digest will inactivated the enzymatic properties. However not all enzyme are inactivated by heat.
Just the list here
http://www.neb.com/nebecomm/products/category1.asp?#2

 

[MusicMonkey]

What is the charge of DNA? In what direction would the DNA moved in your gel under normal conditions?

The charge of DNA is negative. In normal conditions the more negative would move towards the positive end and the more positive would stay around the negative end. Does this mean that if the electrodes are switched the DNA will move the opposite way? Also, if the electrodes are changed after 5 minutes then would it just correct the problem?

It has to do with size of DNA fragment and matrix. 0.8% is usually the gel use the separate a wide diversity of bands. A 2% is to separate smaller fragments and will give a better resolution for small fragments.

Therefore, if there is a very small fragment size and a higher resolution is needed then the 2% will be used.

Increasing the temperature during restriction enzyme digest will inactivated the enzymatic properties. However not all enzyme are inactivated by heat.

But what will happen to the bands appearance on the gel?

Also if lambda DNA is and EcoR1 are reacted together, incubated for one hour at room temperature, 37C and 60C what would be expected when they are electrophoresed?

Would it be correct to say that at 60C the EcoR1 would be inactivated and the DNA would begin to anneal, at 37C there would be no difference, and at room temperature the pairing of the bonds would be unstable and would not last long? If this is correct what would be the expected appearance of the bands on the gel?

Thank you for your continuing help.

 

[iansmith]

The charge of DNA is negative.

That's right

In normal conditions the more negative would move towards the positive end and the more positive would stay around the negative end.

In agarose gel, you separate the DNA according to it size not according to its charge. The smaller size move faster than the larger band. So the larger DNA fragments will be closer to the negative charge.

Does this mean that if the electrodes are switched the DNA will move the opposite way? Also, if the electrodes are changed after 5 minutes then would it just correct the problem?

Yes, the DNA will move the opposite direction and eventually out of the gel but still towards the positive side. What happen is because you separate according to size, the smaller band will have move "higher" (relative to you looking at normal gel picture) than the larger band. Therefore the smaller band do not start at the same position as the larger band and will have to more migration to get to their expected point. This can give you incorrect results if you have several band of approximetly the same size within a certain range (50 bp to about 2 kb). In pratice, reversing the polarity will fix the problem if you only have one band of 500 bp or several bands larger than 3 kb it does not create a problem and not extensive damage will be done. Although, I would not suggest that you publish that gel in any type of publication.

Therefore, if there is a very small fragment size and a higher resolution is needed then the 2% will be used.

It is partly correct. It is more like if you want to differentiate between several small size fragments that have approximetly the same size. For example 5 bands that are between 200 bp and 350 bp. RFLP (Restriction Fragment Length Polymorphism) is a techniques that used higher agarose gel percentage.

But what will happen to the bands appearance on the gel?

Also if lambda DNA is and EcoR1 are reacted together, incubated for one hour at room temperature, 37C and 60C what would be expected when they are electrophoresed?

Would it be correct to say that at 60C the EcoR1 would be inactivated and the DNA would begin to anneal, at 37C there would be no difference, and at room temperature the pairing of the bonds would be unstable and would not last long? If this is correct what would be the expected appearance of the bands on the gel?

So we known that the restriction enzyme that you mention have a peek of activity at 37C. Some become inative at 60C. Do you rememeber how enzyme kinetics are effected by temperature? It should of been seen in a biochemistry course.

Lets assume that at 37 for 2 hours the DNA, a 3 Kb plasmid that is cut by each enzyme 3 times, is fully digested. This will give you a certain parttern on your gels. Knowing that temperature affect activity. Do you expect the plasmid to be fully digested in 2 hours at room temperature (RT) and at 60C.

Edit: So at 37C, there will be 4 bands as the pattern. At RT and 60 there will be an extra band at 3 kb because the enzyme will not fully cut the plasmid due to sub-optimal temperature. In some cases (i.e. EcoR1) if not all, the 3 kb band might be the only visible band because the enzyme activity was not sufficient to cut the DNA or the level that is visible on a gel. At RT, there should be 5 bands; however the band below 3 kb will be at a lower intensity compare to the one at 37C.

You can look at this article if can find it at your library
http://www.ejbiochem.org/cgi/content/abstract/123/1/141

 

[MusicMonkey]

Is this a correct explanation to the following question?
Why is it important to use purified DNA samples in restriction digests and gel electrophoresis (i.e. rather than just using homogenized tissues)?
It is important to use purified DNA samples because if purified DNA samples are not used it will cause the restriction enzymes to cut at incorrect sequences causing improper resulting fragment sizes.

Thank you for your continuing help.

 

[iansmith]

Is this a correct explanation to the following question?
Why is it important to use purified DNA samples in restriction digests and gel electrophoresis (i.e. rather than just using homogenized tissues)?
It is important to use purified DNA samples because if purified DNA samples are not used it will cause the restriction enzymes to cut at incorrect sequences causing improper resulting fragment sizes.

It is partly correct. Homogenized tissues contain proteins that bind to DNA. The protein will, therefore, retard the the migration of the DNA inside an agarose gel. It will also protect the DNA from the restriction enzyme (see method for DNA footprinting) and the restriction enzyme will not cut at every possible site.
There might also be problem with the condition of the restriction digest. Material in the homogenized tissue might interrefered with the restriction enzyme and the condition (such as the pH and ions) of the solution might be sub-optimun. Therefore, the enzyme might not cut efficiently and fewer site will be cut and incorrect site will be cut if the restriction enzyme has been shown to produce star activity (http://www.biochem.northwestern.edu/holmgren/Glossary/Definitions/Def-S/star_activity.html)