Matt:LabNotes/2017-6-27: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (Created page with "=Image and Seq= http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm <br> http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm *Use new acrylamide primer *Anne...") |
>Mzcai m (→Conclusion) |
||
(3 intermediate revisions by the same user not shown) | |||
Line 4: | Line 4: | ||
http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm | http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm | ||
*Use new acrylamide primer | *[[Matt:LabNotes/2017-6-14|Last time]] | ||
*Use new acrylamide primer (needs to be stored in -20C, aliquoted to avoid freeze/thaw) | |||
*Annealing temp of PCR changed from 55C -> 45C | *Annealing temp of PCR changed from 55C -> 45C | ||
*One sample placed on magnet for 1min | *One sample placed on magnet for 1min | ||
*One sample no magnet | *One sample no magnet | ||
*K | |||
==Prepare Beads== | ==Prepare Beads== | ||
Line 39: | Line 41: | ||
==Gel PCR== | ==Gel PCR== | ||
#Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance | |||
#Let gel slide dry in AirClean hood for 30min | |||
#Prepare 25ul PCR mix per gel | #Prepare 25ul PCR mix per gel | ||
#*2.5ul 5uM Primer2 | #*2.5ul 5uM Primer2 | ||
#*12.5ul KAPA SYBR FAST MM | #*12.5ul KAPA SYBR FAST MM | ||
#* | #*5ul beads (1:1 mix of v1 and v3) | ||
#*1.25ul USER | |||
#*3.75ul H2O | |||
#Pipet 25ul onto center of gel | #Pipet 25ul onto center of gel | ||
#Apply 18x30mm cover slip | #Apply 18x30mm cover slip | ||
Line 53: | Line 56: | ||
#Slide PCR on Biorad thermocycler | #Slide PCR on Biorad thermocycler | ||
94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold | 37C 15min -> 94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold | ||
#Place directly on Olympus with GFP/FITC filter | #Place directly on Olympus with GFP/FITC filter | ||
== | ==Results== | ||
===Control (no magnet)=== | |||
[[File:Composite_Control_Pos1.jpg|450px]][[File:Composite_Control_Pos2.jpg|450px]] | |||
== | ===Magnet=== | ||
[[File:Composite_Magnet_Pos1.jpg|450px]][[File:Composite_Magnet_Pos2.jpg|450px]] | |||
*Just GFP: [[File:Magnet_Pos2_GFP.jpg|450px]] | |||
===Special Note=== | |||
*The z plane with fluorescence in focus is not the same as when beads are in focus | |||
*In these images, the in focus BF bead images was merged with in focus fluorescent images | |||
*What the BF channel looks like when the GFP channel is in focus: | |||
[[File:Composite_Magnet_Pos3.jpg|450px]] | |||
[[File: | |||
=== | |||
* | |||
* | |||
* | |||
==Conclusion== | ==Conclusion== | ||
* | *Probably not rolonies, all fluorescent signal has similar characteristics that I have seen in previous tries: | ||
**Bright star shapes that look kind of like cell membrane stretched over cytoskeleton | |||
**Sometimes the star is surrounded by a fainter round signal | |||
* | |||
* | |||
* | |||
* | |||
*Do No Bead Control to see if crap is still here | |||
*Vary bead concentration | |||
*Dr. Zhang says 50-100um polonies is very normal size | |||
**Smaller template has larger polonies because it can diffuse easier | |||
*Do 50+ cycles overnight | |||
Latest revision as of 02:17, 12 July 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
- Last time
- Use new acrylamide primer (needs to be stored in -20C, aliquoted to avoid freeze/thaw)
- Annealing temp of PCR changed from 55C -> 45C
- One sample placed on magnet for 1min
- One sample no magnet
- K
Prepare Beads[edit]
- 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
- Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
- Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
- Vortex
- Pull down 1min on magnet
- Remove supernatant
- Combined 15ul buffer with 15ul amplicon
- BarcUv1 500nM (dsDNA PCR product with Uracil prepared by Dan 5.25.2017)
- BarcUv3 500nM (dsDNA PCR product with Uracil prepared by Dan 5.25.2017)
- 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
Cast 10% gels with primer[edit]
- Make Gel Mix
- 12.5ul 199:1 40% A:B mix
- 1ul 10% BSA
- 33.5ul H2O
- 1ul 50uM Acrydite primer
- Stored in -20C
- 1ul 5% TEMED
- 1ul 5% APS
- Add 18ul to oval and cover with coverslip
- Put slides in argon chamber and polymerize 30min
- Shake in H2O 30min
Gel PCR[edit]
- Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
- 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)
- 1.25ul USER
- 3.75ul H2O
- 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.
- Place one slide on magnet for 1min
- Slide PCR on Biorad thermocycler
37C 15min -> 94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
- Place directly on Olympus with GFP/FITC filter
Results[edit]
Control (no magnet)[edit]
File:Composite Control Pos1.jpgFile:Composite Control Pos2.jpg
Magnet[edit]
File:Composite Magnet Pos1.jpgFile:Composite Magnet Pos2.jpg
- Just GFP: File:Magnet Pos2 GFP.jpg
Special Note[edit]
- The z plane with fluorescence in focus is not the same as when beads are in focus
- In these images, the in focus BF bead images was merged with in focus fluorescent images
- What the BF channel looks like when the GFP channel is in focus:
File:Composite Magnet Pos3.jpg
Conclusion[edit]
- Probably not rolonies, all fluorescent signal has similar characteristics that I have seen in previous tries:
- Bright star shapes that look kind of like cell membrane stretched over cytoskeleton
- Sometimes the star is surrounded by a fainter round signal
- Do No Bead Control to see if crap is still here
- Vary bead concentration
- Dr. Zhang says 50-100um polonies is very normal size
- Smaller template has larger polonies because it can diffuse easier
- Do 50+ cycles overnight