Matt:LabNotes/2015-6-5: Difference between revisions

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===Probe Resuspension===
===Probe Resuspension===
*The 5' amino modified (separated by C6) probes ordered by Dan:
*The 5' amino modified (separated by C6) 48-probe set ordered by Dan:
**GAD1
**GAD1
***Already resuspended
***Already resuspended
Line 13: Line 13:
**SATB2
**SATB2
**KIT
**KIT
*Pick 2-3 genes and [[Daniel:Notebook/RNAFISH/2014-8-30#Oligo_Set_Preparations | resuspend]] if needed
*Pick 2-3 genes and [[Daniel:Notebook/RNAFISH/2015-1-29 | resuspend]] if needed
 
*Dr. Zhang suggested SLC6A1 and SATB2
**[[Media:GAO_022315.xlsx | Table of ordered plates]]
***SLC6A1: Plate=CUX2-SLC6A1-20150128 Wells=E1-H12
***SATB2: Plate=ADARB2-SATB2-20150128 Wells=E1-H12
***Also resuspend CUX2 and ADARB2 for future
**[[Media:RNAFISH_Genes_Info.xlsx | Gene information with CA12kNov14_V4 efficiencies]]
 
#Resuspend each oligo in 15 uL nuclase-free H2O (1 mM)
#Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
##Store the remaining oligos at -20C
#Add 350 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
#*Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower...
#*Goal is to get 1ug/ul DNA concentration and this is what Dan has found to get closest to that
#Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''measured (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''normalized (ng/ul)'''
| align="center" style="background:#f0f0f0;"|'''Average'''
| align="center" style="background:#f0f0f0;"|'''Stdev'''
|-
| ADARB2 1:1||1228||1228||||
|-
| ADARB2 1:5||271.8||1359||||
|-
| ADARB2 1:10||135.4||1354||1313.7||74.2
|-
| CUX2 1:1||1227.6||1227.6||||
|-
| CUX2 1:5||258.5||1292.5||||
|-
| CUX2 1:10||128.8||1288||1269.4||36.2
|-
| SATB2 1:1||1328.8||1328.8||||
|-
| SATB2 1:5||290||1450||||
|-
| SATB2 1:10||138.2||1382||1386.9||60.8
|-
| SLC6A1 1:1||1356.8||1356.8||||
|-
| SLC6A1 1:5||300.3||1501.5||||
|-
| SLC6A1 1:10||147.4||1474||1444.1||76.8
|}
*For ADARB2 & CUX2 with approx 1300ng/ul
**Add 132ul H2O to 440ul
**1300ng/ul x 440ul = 1000ng/ul x 572ul -> 572-440=132ul H2O added
*For SATB2 & SLC6A1 with approx 1400ng/ul
**Add 176ul H2O to 440ul
**1400ng/ul x 440ul = 1000ng/ul x 616ul -> 616-440=176ul H2O added


===Dye Coupling===
===Dye Coupling===
[[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]
[[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br>
[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]]
[[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]]
SLC6A1-488
SATB2-594
#Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
#Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
#Denature samples for 5 minutes at 95C, then snap cool using ice box
#Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
#*Dye cannot be saved for later use.  Use immediately!
#Add 8 uL sample to dye tube
#Incubate in the dark for 1 hour
#Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
#[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation
#*Use 1X TE Buffer
#*No vacufuge
#Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
#Centrifuge at 4 C for 30 minutes (14,000 rpm)
#Remove supernatant and add 750 uL chilled 75% EtOH
#Centrifuge 5 minutes at 4 C
#Dry the pellet in the hood
#Resuspend pellet in 12 uL TE
#Check dye:probe ratio with Nanodrop
#*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
#*Starting DNA mass = 5ul * 1ug/ul
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Probe'''
| align="center" style="background:#f0f0f0;"|'''ssDNA ng/ul'''
| align="center" style="background:#f0f0f0;"|'''488 dye pmol/ul'''
| align="center" style="background:#f0f0f0;"|'''594 dye pmol/ul'''
| align="center" style="background:#f0f0f0;"|'''ssDNA pmol/ul'''
| align="center" style="background:#f0f0f0;"|'''dye:probe ratio'''
| align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)'''
| align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)'''
| align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)'''
|-
| SLC6A1-488||247.8||43.3||0.6||38.18||'''1.13'''||2.97||5||'''59.4'''
|-
| SATB2-594||260.4||-0.6||52.6||40.12||'''1.31'''||3.12||5||'''62.4'''
|}


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Latest revision as of 15:31, 8 June 2015

RNA FISH: Probe Resuspension and Dye Coupling[edit]

Probe Resuspension[edit]

  • The 5' amino modified (separated by C6) 48-probe set ordered by Dan:
    • GAD1
      • Already resuspended
    • FOXP2
      • Already resuspended
    • CUX2
      • Already resuspended
    • SLC6A1
    • ADARB2
    • SATB2
    • KIT
  • Pick 2-3 genes and resuspend if needed
  1. Resuspend each oligo in 15 uL nuclase-free H2O (1 mM)
  2. Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM)
    1. Store the remaining oligos at -20C
  3. Add 350 uL nuclase-free, diluting 10:1, for final concentration of 100 uM
    • Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower...
    • Goal is to get 1ug/ul DNA concentration and this is what Dan has found to get closest to that
  4. Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy
' measured (ng/ul) normalized (ng/ul) Average Stdev
ADARB2 1:1 1228 1228
ADARB2 1:5 271.8 1359
ADARB2 1:10 135.4 1354 1313.7 74.2
CUX2 1:1 1227.6 1227.6
CUX2 1:5 258.5 1292.5
CUX2 1:10 128.8 1288 1269.4 36.2
SATB2 1:1 1328.8 1328.8
SATB2 1:5 290 1450
SATB2 1:10 138.2 1382 1386.9 60.8
SLC6A1 1:1 1356.8 1356.8
SLC6A1 1:5 300.3 1501.5
SLC6A1 1:10 147.4 1474 1444.1 76.8
  • For ADARB2 & CUX2 with approx 1300ng/ul
    • Add 132ul H2O to 440ul
    • 1300ng/ul x 440ul = 1000ng/ul x 572ul -> 572-440=132ul H2O added
  • For SATB2 & SLC6A1 with approx 1400ng/ul
    • Add 176ul H2O to 440ul
    • 1400ng/ul x 440ul = 1000ng/ul x 616ul -> 616-440=176ul H2O added

Dye Coupling[edit]

Dan's best practice dye coupling protocol
Generic dye coupling protocol SLC6A1-488 SATB2-594

  1. Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature
  2. Add 5 uL DNA and 3 uL of sodium bicarbonate buffer
  3. Denature samples for 5 minutes at 95C, then snap cool using ice box
  4. Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing
    • Dye cannot be saved for later use. Use immediately!
  5. Add 8 uL sample to dye tube
  6. Incubate in the dark for 1 hour
  7. Add 10 uL 3M NaOAc and 80 uL 1xTE to sample
  8. Centri-Sep column purification after incubation
    • Use 1X TE Buffer
    • No vacufuge
  9. Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight
  1. Centrifuge at 4 C for 30 minutes (14,000 rpm)
  2. Remove supernatant and add 750 uL chilled 75% EtOH
  3. Centrifuge 5 minutes at 4 C
  4. Dry the pellet in the hood
  5. Resuspend pellet in 12 uL TE
  6. Check dye:probe ratio with Nanodrop
    • Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer)
    • Starting DNA mass = 5ul * 1ug/ul
Probe ssDNA ng/ul 488 dye pmol/ul 594 dye pmol/ul ssDNA pmol/ul dye:probe ratio ssDNA mass (ug) ssDNA start mass (ug) ssDNA Yield (%)
SLC6A1-488 247.8 43.3 0.6 38.18 1.13 2.97 5 59.4
SATB2-594 260.4 -0.6 52.6 40.12 1.31 3.12 5 62.4