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Scientific paper on DNA replication & gel electrophoresis

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Scientific paper on DNA replication & gel electrophoresis

Postby dekk1525 » Sat Sep 08, 2012 11:17 pm

I have to give a short presentation on how the techniques in a scientific paper relate to the techniques that we are doing in lab. I'm concentrating on gel electrophoresis. This article is one of the few I could find that was recent enough and used gel el. The problem is I'm not used to reading scientific papers so I need to make sure I understand what is happening in the article. The article is here. I skipped over anything that used 2D electrophoresis.

To my understanding this is what happened. Please correct anything I have wrong:

-The purpose of the experiment was to study what happens when an E. coli replisome encounters a cyclobutane pyrimidine dimer (CPD) in leading strand of DNA
-They constructed a plasmid containing oriC and a single CPD in the leading-strand
-They inserted two terB sites to get a one unidirectional replication system
-Only the minimal protein required to sustain oriC and replication were used. Topoisomerase was not used to stall replication after a few minutes
-Used Eco RI to cut the DNA
-Mapped the bp lengths of the newly replicated strand every 1 minute for 6 minutes. Strands that migrated to the top of the gel indicate a stalled replication fork. This was the bulk reaction.
-Then used gel-filtration chromatography to remove all unbound proteins and then readded only the 3 proteins that were known to be necessary during replication. The same procedure using gel electrophoresis from before was preformed. This was the column isolated reaction. Gel migrations from the bulk reaction and column-isolated reactions were almost indistinguishable. This suggests that the oriC-loaded replisome can catalyze their formation without the need to recruit additional DNA polymerase or helicase.

What I don't understand:

1. Why are the stalled forks about 23 kbp while the full length DNA are only 9.4 kbp (on figure 1)?
2. Did they prepare 1 sample and run it through the gel every minute for 6 minutes; or did they prepare 1 sample and run it through after 1 minute prepare a different sample and run it through after 2 minutes, etc?
2. What is the purpose of Eco RI is this experiment? I know that it cuts the plasmid at a specific site but I don't know why they did this.


Any help would be appreciated.
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