Matt:LabNotes/2017-6-14: Difference between revisions

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>Mzcai
 
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=Image and Seq=
=Image and Seq=


*[[Last time|Matt:LabNotes/2017-6-13]]
*[[Matt:LabNotes/2017-6-13|Last time]]
**messed up the sample with USER in PCR mix
**messed up the sample with USER in PCR mix
**pre-nicking with USER did not work
**pre-nicking with USER did not work
Line 62: Line 62:


#Place directly on Olympus with GFP/FITC filter
#Place directly on Olympus with GFP/FITC filter
<!--
==Strip Second Strand==
#Remove SecureSeal and put slide in glass Coplin jar of Hexane for 5min
#Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive
#Make Wash 1E: 10mM Tris + 50mM KCl + 2mM EDTA + 0.01% TX-100
#Shake slide in plastic box of Wash 1E twice for 4min
#Apply blue FrameSeal chamber
#*Hoping to use smaller volumes of Wash 1E
#Add ~800ul Wash 1E and then aspirate 2 times


==Strip Second Strand==
==Results==
#Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
*All 3 had deformed SecureSeals and were leaking oil
#Wash twice with Wash 1E
*Used 20X objective to image


==Hybridize FISH probes==
===Sample 1===
#Add 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC preheated to 80C and incubate 10min at room temp
[[File:20170614_Sample1_2ndprimer_composite.jpg|450px]]
#Wash twice with 2X SSC
===Sample 2===
#Add 2X SSC and then seal with glass coverslip
*Spots about 4um in diameter
 
[[File:20170614_Sample2_USER_composite.jpg|450px]][[File:20170614_Sample2_USER1_composite.jpg|450px]]
==Results==
===Sample 3===
===SYBR Green===
[[File:20170614_Sample3_Tween20_composite.jpg|450px]]
[[File:20170604_w2_composite.jpg|650px]]<br>
[[File:20170605_w2_composite.jpg|650px]]<br>
[[File:20170606_w2_composite.jpg|650px]]<br>
===FISH===
[[File:20170607_w1_composite.jpg|650px]]
*Olympus Epifluorescence (GFP in green, Cy5 in red)
**Only saw round signal that could be polony in GFP channel but looked like beads autofluorescence (could also be FISH probes on beads)
**No signal in Cy5 channel (tried exposure time from 100ms to 1s)
*Magnetic beads were still on the gel, often much more clumped together
*No signal under confocal


==Conclusion==
==Conclusion==
*Initially thought the SYBR Green images looked good
*Sample 2 with USER looks promising but could also just be signal similar to [[Matt:LabNotes/2017-6-8#1_Cycle|1 cycle]]
**The fluorescence signal was centered around beads and had diameter of ~8um after 15 cycles
*Sample 1 and 3 are not polonies
**Only a fraction of beads had fluorescence suggesting it was not autofluorescence and true polonies
*Now after cleaning up fluorescent image and overlaying, it seems the fluorescence could be from the bead (maybe SYBR green binding to dsDNA on the bead)
**Fluorescent polony size and BF bead size seem to match perfectly
*No polony signal after FISH, could be due to bad FISH protocol or no polonies...
 
 
*Areas to improve:
*#Cleaner gel (there is too much crap on the gel)
*#*Having a little debris is good for finding focus if there aren't any beads around
*#Image SYBR Green with and without beads
*#*Image right after thermalcycler and then remove beads with magnet and image SYBR Green again
*#Image after varying number of PCR cycles
*#*1 cycle, 15 cycles, 30 cycles
*#To improve FISH, follow the Harvard Protocol more closely


===Harvard Protocol===
*Did some research into acryloyl groups and acrydite-modified primers. Very unstable and should not be stored at 4C like I have been. Order new acrydite-primers and aliquot, store at -20C
#Prepare 70% formamide 1X SSC in plastic coplin jar
*Tm of Primer2 is 53C... so try decreasing annealing temp of PCR to 45C
#*4mL 20X SSC
#*56mL formamide
#*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
-->

Latest revision as of 20:58, 22 June 2017

Image and Seq[edit]

  • Last time
    • messed up the sample with USER in PCR mix
    • pre-nicking with USER did not work

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

Sample Conditions[edit]

3 Samples:

  1. Include 50nM Acrydite primer
  2. Include USER enzyme in the PCR mix
    • USER will nick the biotinylated strand leaving only: CAGTGT
  3. Include 0.1% Tween-20 and 0.2% BSA
    • These are included in the Harvard protocol

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. Wash in H2O 30min shaker

Prepare Beads[edit]

  1. Beads prepared on Matt:LabNotes/2017-5-24

Gel PCR[edit]

  1. Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
  2. Prepare 25ul PCR mix Sample1
    • 2.5ul 5uM Primer2
    • 1.25ul 1uM Acrydite Primer (50nM final)
    • 12.5ul KAPA SYBR FAST MM
    • 5ul beads (1:1 mix of v1 and v3)
    • 3.75ul H2O
  3. Prepare 25ul PCR mix Sample2
    • 2.5ul 5uM Primer2
    • 12.5ul KAPA SYBR FAST MM
    • 1.25ul USER
    • 5ul beads (1:1 mix of v1 and v3)
    • 3.75ul H2O
  4. Prepare 25ul PCR mix Sample3
    • 2.5ul 5uM Primer2
    • 12.5ul KAPA SYBR FAST MM
    • 5ul beads (1:1 mix of v1 and v3)
    • 2.5ul 1% Tween-20
    • 2.5ul 2% BSA
  1. Pipet 25ul onto center of gel
  2. Apply 18x30mm cover slip
  3. Apply an orange SecureSeal chamber
  4. Fill chamber with mineral oil and seal holes with stickies.
  5. Slide PCR on Biorad thermocycler
    • Put in sample 2 first, put in sample 1 & 3 after 37C 30min incubation
 37C 30min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold
  1. Place directly on Olympus with GFP/FITC filter

Results[edit]

  • All 3 had deformed SecureSeals and were leaking oil
  • Used 20X objective to image

Sample 1[edit]

File:20170614 Sample1 2ndprimer composite.jpg

Sample 2[edit]

  • Spots about 4um in diameter

File:20170614 Sample2 USER composite.jpgFile:20170614 Sample2 USER1 composite.jpg

Sample 3[edit]

File:20170614 Sample3 Tween20 composite.jpg

Conclusion[edit]

  • Sample 2 with USER looks promising but could also just be signal similar to 1 cycle
  • Sample 1 and 3 are not polonies
  • Did some research into acryloyl groups and acrydite-modified primers. Very unstable and should not be stored at 4C like I have been. Order new acrydite-primers and aliquot, store at -20C
  • Tm of Primer2 is 53C... so try decreasing annealing temp of PCR to 45C