Lab 4 Flashcards
Alkaline Miniprep
DNA is separated from cellular DNA, RNA, and protein by a series of selective precipitations and extractions
minipreps are used for…
e.coli and gram-negative bacteria
Because the plasmid is a circular molecule…
the DNA strands are twisted around each other and the alkaline environment doesn’t cause complete denaturation and strand separation
SDS and sodium hydroxide are used to…
dissolve the cell membranes, denature proteins, and denature DNA
QiaGen minipreps work best when…
the plasmid is less than 20,000 bp in size
If the sample is pure…
spectrophotometric measurement of the amount of UV irradiation absorbed by the bases is simple and accurate
If the amount of DNA or RNA is very small or the sample contains impurities
the amount of nucleic acid can be estimated from the intensity of fluorescence emitted by ethidium bromide
Isolation and purification of plasmid DNA
take E.coli in LB medium into a BD falcon tube, centrifuge for 5 minutes, discard supernatant (do this twice for PUC18), resuspend in P1 buffer, add P2 buffer and mix. add N3 buffer and mix, centrifuge for 10 minutes, move supernatants to the spin column, centrifuge for 1 minute, discard the liquid, add PE buffer and centrifuge for 1 minute, discard the liquid, centrifuge again with nothing added, discard the bottom of the spin column and place into a microcentrifuge tube, add water to the center of the column and spin for 1 minute
P1 buffer
a resuspension buffer
P2 buffer
lysis buffer solution
N3 Buffer
sets up the binding conditions
PB buffer
enables efficient binding of products to the spin-column membrane
PE buffer
wash buffer to remove contaminants
EB buffer
elution buffer
LyseBlue
prevents errors that lead to inefficient cell lysis and incomplete precipitation of SDS, cell debris, and genomic DNA
