Matt:LabNotes/2016-3-14: Difference between revisions
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>Mzcai |
>Mzcai m (→Day 1) |
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#*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O | #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O | ||
#Wash with nf-1X PBS three times '''and check for degradation''' | #Wash with nf-1X PBS three times '''and check for degradation''' | ||
#Add | #Add 75ul gel casting mix '''filtered and degassed''' | ||
#Cover with plastic coverslips cut and glued together such that it leaves ~0. | #*4% PFA, 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED | ||
#Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm space for gel height | |||
#Seal in plastic bag and vacuum out air before filling with argon | #Seal in plastic bag and vacuum out air before filling with argon | ||
#Let sit at RT for 30min | #Let sit at RT for 30min | ||
#Aspirate non-polymerized gel and wash once with cold 1X SSPE | #Aspirate non-polymerized gel and wash once with cold 1X SSPE | ||
#* | #*Edges did not polymerize but the grey matter is fully covered | ||
#Prepare Reverse Transcription Mix '''on ice''' and add | #Prepare Reverse Transcription Mix '''on ice''' and add | ||
{| {{table}} | {| {{table}} |
Revision as of 00:30, 15 March 2016
DARTFISH on BA8 Sections with PA gel
- Last time using gel to cover section it swelled during RT incubation
- To prevent swelling going to use 4% formaldehyde in the gel solution, which crosslinks the hydrogel to Vectabond treated glass (amine functional groups)
- Also add hydrogel after permeabilization because I think last time the hydrogel prevented effective permeabilization
- Used 4th best section from 1/19/16 BA8 patient 1568 from Yun
- 4% Formaldehyde for fixation is one week old and has been sitting at RT under argon gas
- From <http://swehsc.pharmacy.arizona.edu/sites/swehsc.pharmacy.arizona.edu/files/docs/ci/formaldehyde.pdf>
- "We recommend that 10% buffered formalin solutions be used no longer than 3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5."
Protocol
Day 1
- Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Used 4% PFA from 3/8/2016
- Take out second best remaining BA8 sections (4th from back) from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with cold nf-H2O three times and check for degradation
- Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nf-1X PBS three times and check for degradation
- Add 75ul gel casting mix filtered and degassed
- 4% PFA, 10% Acrylamide/Bis-Acrylamide (199:1), 0.2% BSA, 0.1% APS, 0.1% TEMED
- Cover with plastic coverslips cut and glued together such that it leaves ~0.25mm space for gel height
- Seal in plastic bag and vacuum out air before filling with argon
- Let sit at RT for 30min
- Aspirate non-polymerized gel and wash once with cold 1X SSPE
- Edges did not polymerize but the grey matter is fully covered
- Prepare Reverse Transcription Mix on ice and add
Components | Volume |
H2O | 159 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM N9 | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C