Matt:LabNotes/2017-6-13: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
No edit summary
 
(4 intermediate revisions by the same user not shown)
Line 1: Line 1:
=Image and Seq=
=Image and Seq=


*[[Last time|Matt:LabNotes/2017-6-8]] confirmed no polonies
*[[Matt:LabNotes/2017-6-8|Last time]] confirmed no polonies
**Ideas for why:
**Ideas for why:
***Gels were cast and stored poorly in water (UV slides beforehand, use immediately or store in buffer with EDTA)
***Gels were cast and stored poorly in water (UV slides beforehand, use immediately or store in buffer with EDTA)
Line 41: Line 41:
*Put on cold block 5min
*Put on cold block 5min
*Pull down beads, remove supernatant, and resuspend in 5ul Dynabeads buffer
*Pull down beads, remove supernatant, and resuspend in 5ul Dynabeads buffer
**Was not able to pull down beads so just pipetted the reaction mix (1/5 diluted beads)


==Gel PCR==
==Gel PCR==
Line 58: Line 59:
#*Put in sample 3 first, put in sample 1 & 2 after 37C 30min incubation
#*Put in sample 3 first, put in sample 1 & 2 after 37C 30min incubation
    
    
   37C 30min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
   37C 15min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold
 
*Made a mistake and did 15sec 37C at first, didn't catch the mistake until after 94C 3min incubation probably killing the USER enzyme
**Restarted the thermalcycle program anyway just in case but probably won't work


#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==
#Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
#Wash twice with Wash 1E
==Hybridize FISH probes==
#Add 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC preheated to 80C and incubate 10min at room temp
#Wash twice with 2X SSC
#Add 2X SSC and then seal with glass coverslip


==Results==
==Results==
===SYBR Green===
===Sample 1: Standard===
[[File:20170604_w2_composite.jpg|650px]]<br>
*Like seen before, amplification on beads only
[[File:20170605_w2_composite.jpg|650px]]<br>
[[File:20160613_Sample1_Standard_composite.jpg|450px]]
[[File:20170606_w2_composite.jpg|650px]]<br>
===Samples 2: Prenick with USER===
===FISH===
*Signal only in beads
[[File:20170607_w1_composite.jpg|650px]]
[[File:20160613_Sample2_PreUSER2_composite.jpg|450px]][[File:20160613_Sample2_PreUSER5_composite.jpg|450px]]
*Olympus Epifluorescence (GFP in green, Cy5 in red)
===Sample 3: USER in PCR===
**Only saw round signal that could be polony in GFP channel but looked like beads autofluorescence (could also be FISH probes on beads)
*Again signal only in beads
**No signal in Cy5 channel (tried exposure time from 100ms to 1s)
*Expected because not enough time for USER digest
*Magnetic beads were still on the gel, often much more clumped together
[[File:20160613_Sample3_USER_composite.jpg|450px]][[File:20160613_Sample3_USER1_composite.jpg|450px]][[450px]]
*No signal under confocal
 
==Conclusion==
==Conclusion==
*Initially thought the SYBR Green images looked good
*Need to try USER in PCR again
**The fluorescence signal was centered around beads and had diameter of ~8um after 15 cycles
*Try adding 1/10th free floating acrydite primer
**Only a fraction of beads had fluorescence suggesting it was not autofluorescence and true polonies
*Try following Harvard protocol more closely
*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)
*Fresh gels made no difference
**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===
#Prepare 70% formamide 1X SSC in plastic coplin jar
#*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 00:15, 16 June 2017

Image and Seq[edit]

  • Last time confirmed no polonies
    • Ideas for why:
      • Gels were cast and stored poorly in water (UV slides beforehand, use immediately or store in buffer with EDTA)
      • Primers on beads outcompeted primers in gel (USER digest amplicons on beads to shorten primer on beads)

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 to test how USER can help create polonies
    • USER will nick the biotinylated strand leaving only: CAGTGT

Samples:

  1. Basic repeat of last time with fresh gels
  2. USER digest beads before adding to gel
  3. Include USER enzyme in the PCR mix

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

For USER prenicked Beads (Sample 2)[edit]

  • Take 5ul beads (1:1 mix of v1 and v3)
  • Add 2.5ul 10X Cutsmart Buffer
  • Add 2.5ul USER
  • Add 15ul H2O
  • Incubate 15min @ 37C
  • Put on cold block 5min
  • Pull down beads, remove supernatant, and resuspend in 5ul Dynabeads buffer
    • Was not able to pull down beads so just pipetted the reaction mix (1/5 diluted beads)

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 per gel
    • 2.5ul 5uM Primer2
    • 12.5ul KAPA SYBR FAST MM
    • 5ul beads (1:1 mix of v1 and v3)
    • 5ul H2O
    • For USER sample 3 include 1.25ul USER and use 3.75ul H2O instead
  3. Pipet 25ul onto center of gel
  1. Apply 18x30mm cover slip
  2. Apply an orange SecureSeal chamber
  3. Fill chamber with mineral oil and seal holes with stickies.
  4. Slide PCR on Biorad thermocycler
    • Put in sample 3 first, put in sample 1 & 2 after 37C 30min incubation
 37C 15min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold
  • Made a mistake and did 15sec 37C at first, didn't catch the mistake until after 94C 3min incubation probably killing the USER enzyme
    • Restarted the thermalcycle program anyway just in case but probably won't work
  1. Place directly on Olympus with GFP/FITC filter

Results[edit]

Sample 1: Standard[edit]

  • Like seen before, amplification on beads only

File:20160613 Sample1 Standard composite.jpg

Samples 2: Prenick with USER[edit]

  • Signal only in beads

File:20160613 Sample2 PreUSER2 composite.jpgFile:20160613 Sample2 PreUSER5 composite.jpg

Sample 3: USER in PCR[edit]

  • Again signal only in beads
  • Expected because not enough time for USER digest

File:20160613 Sample3 USER composite.jpgFile:20160613 Sample3 USER1 composite.jpg450px

Conclusion[edit]

  • Need to try USER in PCR again
  • Try adding 1/10th free floating acrydite primer
  • Try following Harvard protocol more closely
  • Fresh gels made no difference