Lab 8

Question 1

What type of solution favors plasmid DNA binding to the “ Qiaprep” silica gel spin column (e.g, in the combination solution P1+P2+N3), and what favors release( e.g, solution EB)?

Answer 1

high salt is DNA binds to the silca gel

low salt is DNA comes back off silica

Question 2

What can we learn the gel electrophoresis band result obtained with our plasmid minipreps at the end of the last lab (sample below) ; can the gel lanes shown below help us decide which plamid prep to send or perhaps not to send for DNA sequenceing ?

Answer 2

DNA yield and size

the fast moving plasmid are large and supercoiled which are notarized to be sent to be sequencing

Question 3

We will use gel electrophoresis to analyze EcoRl-cut plasmid minprep DNA, to compare to the uncut (intact) plasmid miniprep DNA analyzed at the end of the last lab What relative electrophoretic profiles should we expect when comparing results with intact, uncut plasmid DNA ,versus ECorl-cut plasmid DNA ?

Answer 3

uncut supercoil gel - one main plasmid (supercoil ) band lighter nicked circle band

Ecorl-cut 2 band, vector + insert is varies
3kb

Question 4

Following cell lysis, we quenched the lysis conditions by adding Qiagen buffer N3, and then obtained a precipitate. What was in this precipitate and what was not ?

Answer 4

Precipitate- E.coil genomic DNA

Supernatant - contains plasmid DNA

Question 4

What is the chemical basis for lysis of the E.coil cells, i.e,. what the the two critical compoents of buffer P2 ?

Answer 4

Alkaline ph +SDS

Question 5

What would be the expectd relative agarose gel electrophoretic band mobility of a particular plasmid DNA molecule(e.g a 5 kb DNA) when it is in the following physical form:
i) covalently closed circular and supercoild. ii) nicked open circular or iii) linear ?

Answer 5

covalently closed circular and supercoild is fastest moving

nicked open circular is slowest moving

linear between (i) and (ii)