MIT Department of Biology
7.02 Experimental Biology & Communication, Spring 2005
Module overview
Goal
• Zebrafish
development
observation
Technique
• Phase contrast
microscopy
• Teratogenesis
• Gene expression • RNA isolation
analysis
• Northern blot
TODAY
TODAY
Northern blot: steps
Run Denaturing
Agarose Gel
Transfer to Nylon
Membrane
Fix RNA on the Membrane and Prehybridize
Hybridize with Labeled
DNA Probes
Detect Probe on a Film
Figures by MIT OCW.
1
Today’s topics
• Transfer
• RNA fixation
• Probe labeling
How is RNA/DNA transferred?
DRY paper towels
• Through capillary
action.
• This is possible
because RNA and
DNA are soluble at
PH 7.
membrane
gel
buffer
2
Transfer apparatus
• Whatman paper wick (ends immersed in buffer)
• Gel (inverted; EVEN side in touch with membrane)
• Nylon membrane
• 1 wet Whatman paper
• 2 dry Whatman paper
• A stack of DRY paper towels
UV crosslinking
• Purpose: fix RNA/DNA on the membrane.
• Reason: RNA and DNA are soluble.
U
NH2 U
U
NH2 NH2
U
U
U
NH2 U NH2
U
U
NH2
NH2
U NH2
NH2
NH2
U
• Don’t want to overcrosslink
hybridization efficiency.
decreased
3
Probe labeling
• Probe: z-cyt1 cDNA (complimentary to mRNA)
• Label: digoxingenin on dUTP
• Labeling method: digoxingenin-dUTP is
incorporated into DNA probes by random
priming.
• Random priming: using random hexanucleotide
primers that are not gene specific.
Random priming
Reaction mix:
DNA template,
random hexanucleotides,
dNTP (100µM dATP, dCTP and dGTP; 65 µM dTTP),
DIG-dUTP (35 µM),
Klenow DNA polymerase,
and buffer
4
Random priming
dsDNA template
95°C denature
37°C anneal
37°C extension
Probe labeling vs PCR
template
Z-cyt1 cDNA
1µg
Template amount
Denature T (°C )
95
#Primers
46(in theory)
Primer size
6
Annealing T (°C )
37
Extension T (°C )
37
Klenow
enzyme
dNTPs
DIG-dUTP
1
#cycles
Genomic DNA
20ng
95
2
20
55
72
Taq
dNTP
30
5