Matt:LabNotes/2017-5-24: Difference between revisions

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>Mzcai
(Created page with "=Image and Seq= http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm ==Cast 5% gels with primer== 25ul ABD mix 2ul 10% BSA 67ul H2O 2ul 50uM primer 2ul 5% TEMED 2ul...")
 
>Mzcai
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http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm
http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm
http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm


==Cast 5% gels with primer==
==Cast 10% gels with primer==
#Make Gel Mix
#*25ul 199:1 40% A:B mix
#*2ul 10% BSA
#*67ul H2O
#*2ul 50uM Acrydite primer
#*2ul 5% TEMED
#*2ul 5% APS
#Add 18ul to oval and cover with coverslip
#Put slides in argon chamber and polymerize 30min
#Put in MilliQ H2O overnight
#*Normally Wash in H2O 30min shaker


25ul ABD mix
==Prepare Beads==
2ul 10% BSA
#Prepare 50ml Dynabeads buffer: 20ml 5M NaCl + 100ul 0.5M EDTA + 125ul 4M Tris-HCl + 29.775ml H2O
67ul H2O
#*2M NaCl, 1mM EDTA, 10mM Tris-HCl
2ul 50uM primer
#Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
2ul 5% TEMED
#Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
2ul 5% APS
#Vortex
#Pull down 1min on magnet
#Remove supernatant
#Combined 15ul buffer with 15ul amplicon
#*BarcUv1 240nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
#*BarcUv3 220nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
#Add to beads, pipette mix, and incubate 15min at room temp
#Pull down
#Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet)
#Bring volume to 40ul with H2O
 
==Gel PCR==
  Prepare diffuse-in mix while allowing slides to dry.  You don't want the slides to over-dry, though.  You will observe a thin, shrinking film of liquid on the surface of each gel.  Generally, we try to add the diffuse-in mix within 5 minutes or so of this shrinking film's disappearance.  This generally means about 30 minutes of drying.


Add 18ul to oval and cover with coverslip
#Let gel slide dry in AirClean hood for 30min
Put slides in argon chamber and polymerize 30min
#Prepare 25ul PCR mix per gel
Put in MilliQ H2O overnight
#*2.5ul 5uM Primer2
**Normally Wash in H2O 30min shaker
#*12.5ul KAPA SYBR FAST MM
#*10ul beads (1:1 mix of v1 and v3)
#Pipet 25ul onto center of gel
#Apply 18x30mm cover slip
#Apply an orange SecureSeal chamber
#Fill chamber with mineral oil and seal holes with stickies.
#Slide PCR on Biorad thermocycler
 
  94C 3min -> (94C 45sec -> 55C 30sec -> 72C 1min) x 7 -> 72C 3min -> 4C hold


==Prepare Beads==
#Remove SecureSeal and put slide in glass Coplin jar of Hexane for 10min (should be 5min)
*Prepare 50ml Dynabeads buffer: 20ml 5M NaCl + 100ul 0.5M EDTA + 125ul 4M Tris-HCl + 29.775ml H2O
#Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive
**2M NaCl, 1mM EDTA, 10mM Tris-HCl
#Make Wash 1E: 10mM Tris + 50mM KCl + 2mM EDTA + 0.01% TX-100
*Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
#Shake slide in Coplin jar of Wash 1E twice for 4min
*Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
#Store in Wash 1E
*Vortex
*Pull down 1min on magnet
*Remove supernatant
*Combined 15ul buffer with 15ul amplicon
**BarcUv1 240nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
**BarcUv3 220nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
*Add to beads, pipette mix, and incubate 15min at room temp
*Pull down
*Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet)
*Bring volume to 40ul with H2O


==Gel PCR==
==Strip Second Strand==
Prepare diffuse-in mix while allowing slides to dry.  You don't want the slides to over-dry, though.  You will observe a thin, shrinking film of liquid on the surface of each gel.  Generally, we try to add the diffuse-in mix within 5 minutes or so of this shrinking film's disappearance.  This generally means about 30 minutes of drying.  
  At the conclusion of the PCR reaction, amplicons are present in double-stranded form, with one strand anchored to the gel via the acrydite modificationDenaturation will permit us to remove the un-anchored strand, such that the remaining strand can be free to serve as a template for hybridization and sequencing reactions.


Prepare 25ul PCR mix per gel
#Apply blue FrameSeal chamber
*2.5ul 5uM Primer2
#Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
*12.5ul KAPA SYBR FAST MM
#Wash twice with 30% formamide in 2X SSC
*10ul beads
#Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
#Wash twice with 30% formamide in 2X SSC


*Pipet 25ul onto center of gel
===Harvard Protocol===
*Apply 18x30mm cover slip
#Prepare 70% formamide 1X SSC in plastic coplin jar
*Apply an orange SecureSeal chamber
#*4mL 20X SSC
*Fill chamber with mineral oil.  This is a little tricky but you get good with practice.  Use a P1000 pipetman filled with ~550 uL of mineral oil.  Use a Chemwipe to dry off the tip of the pipet tip.  The goal is to get the mineral oil in without spilling any near the surface of the hole (as this makes it more of a pain when you go to seal the hole with the stickies).  A good technique to avoid spilling is to hold the slide tilted at a 45-degree angle while filling.  Use the lower hole (in whatever direction you are holding the slide) to fill.  It is OK to leave some air near the upper hole- more important to avoid letting oil seep out of it.  When sufficient oil is in, turn the slide flat (0 degrees) before withdrawing the pipetman from the hole.
#*56mL formamide
*Wipe off any excess mineral oil from the surface of the SecureSeal, if neccessary.  Seal holes with stickies.
#*20mL H2O
#Heat to 70C in microwave (~30sec in microwave) being careful not to let it boil over
#Put slides in and shake in 70C incubator 15min
#Transfer slides to separate coplin jar
#Wash in dH2O 3min on shaker
#Wash twice in Wash 1E 4min on shaker


==FISH==
#Prepare 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC
#Add probes preheated to 75C and incubate 10min at room temp
#Wash with 2X SSC twice
#Add 2X SSC and then cover with coverslip
#Image


  94C 3min -> (94C 45sec -> 55C 30sec -> 72C 1min) x 7 -> 72C 3min -> 4C hold
=Results=
*Didn't see anything under confocal in either channel
*When trying to remove coverslip to stain with SYBR gold the sample got ruined


*Remove SecureSeal and put slide in glass Coplin jar of Hexane for 5min
=Next Time=
*Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive
*Try imaging directly after PCR (using SYBR Green as dye)
*Make Wash 1E: 10mM Tris + 50mM KCl + 2mM EDTA + 0.01% TX-100
**That way can keep increasing number of cycles
*Shake slide in Coplin jar of Wash 1E twice for 4min
*Store in Wash 1E

Latest revision as of 00:16, 31 May 2017

Image and Seq[edit]

http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm

Cast 10% gels with primer[edit]

  1. Make Gel Mix
    • 25ul 199:1 40% A:B mix
    • 2ul 10% BSA
    • 67ul H2O
    • 2ul 50uM Acrydite primer
    • 2ul 5% TEMED
    • 2ul 5% APS
  2. Add 18ul to oval and cover with coverslip
  3. Put slides in argon chamber and polymerize 30min
  4. Put in MilliQ H2O overnight
    • Normally Wash in H2O 30min shaker

Prepare Beads[edit]

  1. Prepare 50ml Dynabeads buffer: 20ml 5M NaCl + 100ul 0.5M EDTA + 125ul 4M Tris-HCl + 29.775ml H2O
    • 2M NaCl, 1mM EDTA, 10mM Tris-HCl
  2. Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
  3. Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
  4. Vortex
  5. Pull down 1min on magnet
  6. Remove supernatant
  7. Combined 15ul buffer with 15ul amplicon
    • BarcUv1 240nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
    • BarcUv3 220nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017)
  8. Add to beads, pipette mix, and incubate 15min at room temp
  9. Pull down
  10. Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet)
  11. Bring volume to 40ul with H2O

Gel PCR[edit]

 Prepare diffuse-in mix while allowing slides to dry.  You don't want the slides to over-dry, though.  You will observe a thin, shrinking film of liquid on the surface of each gel.  Generally, we try to add the diffuse-in mix within 5 minutes or so of this shrinking film's disappearance.  This generally means about 30 minutes of drying. 
  1. Let gel slide dry in AirClean hood for 30min
  2. Prepare 25ul PCR mix per gel
    • 2.5ul 5uM Primer2
    • 12.5ul KAPA SYBR FAST MM
    • 10ul beads (1:1 mix of v1 and v3)
  3. Pipet 25ul onto center of gel
  4. Apply 18x30mm cover slip
  5. Apply an orange SecureSeal chamber
  6. Fill chamber with mineral oil and seal holes with stickies.
  7. Slide PCR on Biorad thermocycler
 94C 3min -> (94C 45sec -> 55C 30sec -> 72C 1min) x 7 -> 72C 3min -> 4C hold
  1. Remove SecureSeal and put slide in glass Coplin jar of Hexane for 10min (should be 5min)
  2. Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive
  3. Make Wash 1E: 10mM Tris + 50mM KCl + 2mM EDTA + 0.01% TX-100
  4. Shake slide in Coplin jar of Wash 1E twice for 4min
  5. Store in Wash 1E

Strip Second Strand[edit]

 At the conclusion of the PCR reaction, amplicons are present in double-stranded form, with one strand anchored to the gel via the acrydite modification.  Denaturation will permit us to remove the un-anchored strand, such that the remaining strand can be free to serve as a template for hybridization and sequencing reactions.
  1. Apply blue FrameSeal chamber
  2. Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
  3. Wash twice with 30% formamide in 2X SSC
  4. Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
  5. Wash twice with 30% formamide in 2X SSC

Harvard Protocol[edit]

  1. Prepare 70% formamide 1X SSC in plastic coplin jar
    • 4mL 20X SSC
    • 56mL formamide
    • 20mL H2O
  2. Heat to 70C in microwave (~30sec in microwave) being careful not to let it boil over
  3. Put slides in and shake in 70C incubator 15min
  4. Transfer slides to separate coplin jar
  5. Wash in dH2O 3min on shaker
  6. Wash twice in Wash 1E 4min on shaker

FISH[edit]

  1. Prepare 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC
  2. Add probes preheated to 75C and incubate 10min at room temp
  3. Wash with 2X SSC twice
  4. Add 2X SSC and then cover with coverslip
  5. Image

Results[edit]

  • Didn't see anything under confocal in either channel
  • When trying to remove coverslip to stain with SYBR gold the sample got ruined

Next Time[edit]

  • Try imaging directly after PCR (using SYBR Green as dye)
    • That way can keep increasing number of cycles