How to keep things RNase free
ice
guanidine thiocyanate
gloves
DEPC
DEPC
binds histidine, tyrosine
guanidine thiocyanate
lyses cells
phenol chloroform
removes protein, organic contaminants
AMV-RT
retrovirus to transcribe cDNA from mRNA
Primers
start transcription during annealing
for a certain amplicon or
if TTTTT selects for mRNAs
dNTPs
nucleotides to synthesize amplicon
TOPO-TA
type I topoisomerase
Kanamycin
Abx that inhibits Tl
Top10 cells
deletion of lacZ operon (lac delta 74) allows for only metabolism of X-gal
LB+KX plates
E coli with plasmid (Kanr) can grow
X-gal
cleaved by beta-gal; differential on plates. white=insert, uncleaved.
blue=whole, cleaved
transformation efficiency
cfu/ml LB+KX x 100
cfu.mL LB
TAE/TBE
tris: pH 8 buffer
ion source: Acetate, Borate
metal chelator: EDTA
Sol'n 1
RNas, Tris, EDTA
Sol'n 2
NaOH inc pH denatures protein and DNA
amphipathic SDS denatures proeins and membranes
Goal: denature cell molecules
Sol'n 3:
KoAc, neutralizes pH
plasmid reconstitutes
bridges DNA backbone and prevents reconstitution
Sol'n 4
EtOH precipitates DNA from salts and organics with Na+ shielding
Sol'n 5
elution buffer-releases DNA from matrix in spin column
Reasons why orientation may differ
cells can grow better in Kanamycin
cells are expressing yeast actin
insertion in coding region->screening
OriZ may allow some LacZalpha activityc