BMD RES 5HA Lecture Notes - Northern Blot, Western Blot, Plasmid
4 May 2018
School
Department
Course
Professor
Deconstruction Lecture 6!•
Looking at the data!•
Exon 2 probe - anything with exon 2 is picked up!◦
her northern blot shows that there are two forms!◦
the ladder showed that the top band met the size of the unspliced form!◦
there were two controls added to the northern blot!◦
gcr1 delta : shows the specificity of the probe, the probe shouldn't bind without gcr, which ◦
it did!
the other control was gcr1+nsDNA !◦
forced a cell to not splice, then she compared the sizes!‣
the lanes weren't horizontal, but it just shows that the electric current wasn't equal ◦
everywhere!
the two controls confirmed that gcr1 probe was specific in grr!◦
the loading control in this northern blot was SCR1!◦
especially in the last lane, the loading control shows that the absence of gcr1 delta ‣
wasn't due to there being no ran!
just because the rna is made, doesn't mean that the protein is made!◦
main idea of the northern: GCR1 RNA changes over time!◦
RNA is made but then she checked if the protein were made!◦
she scanned her northern blot to create a computer generated graph to show the RNA ◦
density!
because the ratio changes over time, she thought that the unspliced version would be ◦
important!
to look for protein expression, she will run proteins through a western blot!‣
through a plasmid, she added different versions of GCR1 !‣
the first one was normal GCR1 called G (genomic dan)!‣
can the cell make the proteins?!•
prediction: will make 2 proteins!•
the next versions!‣
eliminated the branch point: version 2!•
the cell cannot splice, so it is forced to make one type of RNA so one type of •
protein will be made!
prediction: one protein!•
nsDNA (manmade DNA)!•
Regular spliced RNA!‣
version 3, there are no introns!•
prediction: make only one protein!•
ALL versions done in VITRO!‣
loading control shows that they all have the same amount of protein!◦
should be in a cell that lacks GCR1, to not confuse the gcr1 you added with the gcr1 ‣
the cell makes!
make sure all the RNA is recognizable from the species!‣
vector control: DNA carrier (plasmid)!‣
also called an empty vector control!•
ALWAYS must include the empty vector control when doing plasmids!•
vector lane: nothing, what we'd expect!•
the normal RNA lane, shows that there are non spliced versions of the RNA •
being made into proteins!
since the protein is being made, then we know that it must had a reason!◦
we know the un spliced version is made, but how do we know that is it functional!◦
