Midterm 2 Flashcards
DNA isolation -hair follicle reagents/steps
PCR H buffer -100 uL
protease K -2.5 uL of 10 mg/mL
add sample and invert
pulse
incubate at 56 for 1 hour
heat to 95 for 10 min
cool to room temp
PCR buffer H reagents
KCL
Tris HCL - pH 8.3
magnesium chloride
gelatine
NP-40
Tween -20
top off with dH2O until 100 mL
Hair PCR master mix reagents
Dream taq buffer
dNTP
primer 1 and 3
dream taq
dH2O
What temps are used for PCR
denature at 95
anneal at 55-68
elongate at 72
typically 30 seconds each but more for the first and last ( 3 and 5 min respectively
what is colony PCR
add some E.coli directly directly into the PCR and use primers; one to the plasmid back bone immediately to the left and right of the insert and the other that binds to the inset
only amplifies if in the right location
primers must amplify in opposite directions
RNA Isolation from plant tissue
add leaves and homogenize in tripure ( containing guanidinium a chatotropic bassed cell lysis) and phenol
flash freeze in liquid nitrogen to inactivate RNAase with in 1-2 seconds
homogenize again for 1 min
add 1 mL of tripure isolating reagent (TIR)
RNA purification from homogenate
incubate samples for 5 min to ensure disassociation nucleoprotien
add 0.2 mL of chloroform
vortex
incubate at RT for 10-15 min
spin at 12,000 g for 15 min at 4 degrees
transfer colourless aqueous
Chloroform separates the sample into what phase
solution containing lower red
phenol-chloroform interphase
bad the colourless upper aq with the RNA
RNA precipitation and washing
0.5 mL of Isopropanol
cap and invert
incubate at RT for 10 min
spin at 12,000 g for 10 min at 4 degrees
decant supernatant
add cold 75% ethanol gently invert and incubate at -20 for 20 min
spin at 7,500 g for 30 min at 4 degrees disgard supernatant
air dry - but not completely to avoid difficulty in resuspending it
resuspend in 15 uL of DEPC - treated RNA free water
help disolve RNA pellet by gently mixing and incubate for 10-15 min at 55-65 degrees
What gel was teh RNA run on? what are the reagents?
run on 1.2 formaldehyde gel
RNA
denaturing buffer:
-10X MOPS buffer
-formaldehyde
-formamide
-ethidium bromide
RNA blot protocol
Prep membrane - wet in DEPX water and was for 10 seconds in 10X ssc, blot to dry, air dry
Sample Prep - check concentration and calculate volume for 2-3 ug of RNA, heat at 65 degrees for 10 min, return to ice, spin to collect RNA, load in 2 uL aliquots allowing to dry for about 15 min b/w
cross link with UV crosslinker
What is Tripure
a strong denaturing agent that solubilizes tissue and inactivates nucleases
allows for isolation of RNA, DNA, and proteins
What is DEPC
reacts with His residues and inactivates RNAase
carcinogenic and must be autoclaved to ethanol and CO2
if not inactivates it could modify RNA
reacts with tris so solutions with tris buffer must be prepped with DEPC -treated water
makeing RNA formaldehyde gel
1 X MOPA gel running buffer
prep gel
- 1.2 g of agarose
- autoclaved water
- mops
heat in microwave and cool to 60 degrees then add formaldehyde (37 %)
prep samples:
- add 1 volume of RNA to 3 of denaturing buffer
-heat to 65 degrees for 10 min
-ice for 5 min
-spin
How does CRISPR function in bacteria
when first infected the Cas complex bind to the foreign DNA of the phage or the plasmid and insert into the genome of the bacteria as a novel spacer in the CRIPR locus.
once transcribed and processed by Cas II the new, virally derived RNA sequence (crRNA) complexes with another Cas protein which target subsequent foreign DNA
Using CRISPR in eukaryotes
Cas 9 and sgRNA are added to a cell via transformation of a plasmid that contains both.
the sequence of the sgRNA directs the cleavage of the cognate DNA sequence which will eventually lead to NHEJ
specific edits can be made by supplying a DNA template
Steps for heatshocking S/pome
thaw cyopreserved cells
add 1 ug of PCR product and 1 ug od dRNA plasmid
add 50 % PEG 4000 helps DNA adhere to the cell membrane
incubate at 30 degrees for 60 min
heat shock at 43 degrees water bath for 15 m9j\spin for 3 min at 1600 g at RT
decant supernatant and resuspend cells in 1/2 YE medium supplemented with uracil and adenine
incubate at 30 degrees for 30 min while being shaken
mircrofuge for 3 min at 1600 g
decant supernatant and resuspend in TE
spread mixture directly on to EMM2 media contain no uracil and is supplemented with adenine and agar.
prep electro competent S. pome
inoculate LB broth
transfer the cells into a chilled bottle and spin at 18k for 10 min at 4 degrees
discard supernatant and 250 mL of ice cold sterile water
and invert
harvest by spin and wash 3 times with 250 mL of ice cold water
resuspend in 5 mL of 10 % glycerol and again recover with spin
resuspend in 400 uL of 10% glycerol
store at -80 degrees
Details of colony PCR on S.pome
- 5ul of water + colony sample
Master mix:
10x pcr bufferd NTP
ade6 F1 forward primer
OM13 reverse rimer
homemade taq pol
PCR dH2O
Why is SDS used in the formaldehyde gel? How does SDS denature proteins?
Coats the proteins and gives them a negative charge –> (-) is proportional to MW. Also breaks protein-protein bonds –> prevent proteins clumping and skewing their travel
SDS PAGE gel prep
dH2O
1.5 M Tris pH 8.8 (FOR SEPERATING) and 6.8 (STACKING)
40% acrylamide -bisacrylamide
10% SDS
10%APS - is an oxidizing agent that is used with TEMED to catalyze the polymerization of acrylamide and bisacrylamide.
temed - add last then immediately poor filling up to 1 cm bellow the comb
separating and stacking gel differences
-Separating gel uses tris pH 8.8
-Stacking used pH 6.8
-Theproteins hit the interphase (sudden chaneg of pH) –> causes them to slow down “stack up” therefore forming crisp/straight bands
no stacking gel –> wiggly/curved bands (inaccurate analysis)
Add bromanthol blue to stacking gel to help visualize the wells
Coomassie blue staining of the formaldehyde gel
60C stain 10 min
RT destain 30 min
Immunostaining transfer buffer reagents
tris
glycine
SDS
methanol
dH2O
pH 9.2
