Matt:LabNotes/2017-4-12: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
mNo edit summary
>Mzcai
mNo edit summary
 
(12 intermediate revisions by the same user not shown)
Line 1: Line 1:
==Agi15k_Feb2017_V4 Probe Production==
==Agi15k_Feb2017_V4 Probe Production==
*Production of Padlock Probes (V4) [[Matt:LabNotes/2017-2-8]]
*Production of Padlock Probes (V4) [[Matt:LabNotes/2017-2-8]]
===Notes for next time===
*Do 12 or 13 cycles Production PCR
*Experiment with 1hr vs 2hr Lambda digestion
*Think of way to maximize USER and DpnII digestion


===Production PCR===
===Production PCR===
Line 22: Line 27:
| Total||100||10000
| Total||100||10000
|}
|}
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold<br>
[[File:20170412_Agi15kFeb2017_ProductionPCR.png | 600px]]
[[File:20170412_Agi15kFeb2017_ProductionPCR.png | 600px]]
*Do 12 or 13 cycles next time
*Do 12 or 13 cycles next time
Line 45: Line 50:
*Eluted each column with 50ul and combined all into one 1.5ml
*Eluted each column with 50ul and combined all into one 1.5ml
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:
<!--
~400ul of V4 probes: 327.7 ng/ul => ~131 ug
~400ul of V4 probes: 111.1 ng/ul => ~44.4 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
Line 63: Line 67:
|}
|}


*Incubated at 37C for 1hr
*Incubated at 37C for 2hr
*Purified with 4 Zymo ssDNA/RNA columns
*Purified with 8 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**92.9 ng/ul x 160ul 14.9 ug (67% yield)
**95.1 ng/ul x 320ul 30.4 ug (46.5% yield)
***Maybe try less lambda digestion time? or more?


===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====
*Split into 4 PCR tubes and add 5ul USER
*Split into 6 PCR tubes and add 5ul USER
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| SS-amplicon||59
| SS-amplicon||52
|-
|-
| USER||5
| USER||5
Line 82: Line 87:
| 10X DpnII Buffer||8
| 10X DpnII Buffer||8
|-
|-
| H2O||8
| H2O||15
|-
|-
| Total||80
| Total||80
Line 109: Line 114:
*Eluted 20ul each column (80ul total)
*Eluted 20ul each column (80ul total)
*Nanodrop
*Nanodrop
**103.7ng/ul x 80ul = 8.3ug (56% yield)
**114.9ng/ul x 120ul = 13.8ug (% yield)


===PAGE Size Selection===
===PAGE Size Selection===
*Run 2 gels
*Run 4 gels
**200V for 40min
**200V for 40min
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
|-
|-
| V4 Probes||80
| V4 Probes||120
|-
|-
| TBE-Urea Buffer 2X||80
| TBE-Urea Buffer 2X||120
|}
|}


{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
|-
|-
| Low Mass Ladder||2
| Low Mass Ladder||4
|-
|-
| TBE-Urea Buffer 2X||10
| TBE-Urea Buffer 2X||20
|-
|-
| H2O||8
| H2O||16
|-
|-
| Total||20
| Total||40
|}
|}
[[File:2015-05-13_CA12kNov2014_V4_SizeSelect.jpg|450px]]
*Did not save images but could see digestion was incomplete (<1% completely undigested, ~50% half digested)
**Increase USER incubation? Increase DpnII guide oligo concentration?


===EtOH Precipitation===
===EtOH Precipitation===
*Put cut out gel in 4 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Put cut out gel in 8 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
*Transferred the gel remaining in 0.5 mL tube to 2 mL tube below with pipette tip.
*Added 450 ul of 1X TE buffer to each
*Added 450 ul of 1X TE buffer to each
*Vortexed for 60min at 37 C in incubator
*Vortexed for 60min at 37 C in incubator
Line 145: Line 151:
*Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
*Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
*Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
*Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
*Transferred spnt to fresh 1.5 mL tube (~1700ul)
*Transferred spnt to fresh 1.5 mL tube
*Combined with [[Matt:LabNotes/2015-5-5#EtOH_Precipitation_2 | ~850ul from 5/5/2015]]
*Precipitated in 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
*Precipitated in 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
*Vortexed and placed the 6 tubes at -80C overnight
*Vortexed and placed the 6 tubes at -80C for 30min
*Spun 8 tubes at 10,000rpm at 4C for 30min
*Spun 6 tubes at 10,000rpm at 4C for 30min
*Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
*Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
*Discard supernatant and let dry in hood for 10min
*Discard supernatant and let dry in hood for 10min
Line 155: Line 160:


===Qubit Quantification===
===Qubit Quantification===
*22.5 ng/ul => 22.5 ng/ul / (150nt*325/nt + 79Da) = 461nM (60ul)
*10.3 ng/ul => 10.3 ng/ul / (157nt*325/nt + 79Da) = 201.5 nM (80ul)
-->

Latest revision as of 21:53, 18 April 2017

Agi15k_Feb2017_V4 Probe Production[edit]

Notes for next time[edit]

  • Do 12 or 13 cycles Production PCR
  • Experiment with 1hr vs 2hr Lambda digestion
  • Think of way to maximize USER and DpnII digestion

Production PCR[edit]

Components Volume (1X) Volume (100X)
First round amplicon Agi15k_Feb2017_V4 (25nM) 0.1 10
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4910
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
File:20170412 Agi15kFeb2017 ProductionPCR.png

  • Do 12 or 13 cycles next time

EtOH Precipitation[edit]

  • 8 5-ml tubes (with 12 wells of PCR product each) for V4
    • 1200ul PCR product
    • 3000ul 100% EtOH
    • 3ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 10,000rpm at 4C for 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 15 min at 4C
  • Discarded supernatant and air-dried for 10 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification[edit]

  • Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~400ul of V4 probes: 327.7 ng/ul => ~131 ug

Lambda Exonuclease Digestion[edit]

  • Divide into 4 pcr tubes of ~100ul with total amplicon of <10ug each
Components Volume
Amplicon 96
10X Lambda Exo Buffer 12
Lambda Exonuclease 12
Total 120
  • Incubated at 37C for 2hr
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 95.1 ng/ul x 320ul = 30.4 ug (46.5% yield)
      • Maybe try less lambda digestion time? or more?

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 6 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 52
USER 5
10X DpnII Buffer 8
H2O 15
Total 80
  • Incubate at 37C for 2.5 hours

DpnII[edit]

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 2
100uM RE-DpnII_V4 guide oligo 5
H2O 8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (~15hrs)

Zymo Column Purification[edit]

  • Eluted 20ul each column (80ul total)
  • Nanodrop
    • 114.9ng/ul x 120ul = 13.8ug (% yield)

PAGE Size Selection[edit]

  • Run 4 gels
    • 200V for 40min
Components 4X Volume
V4 Probes 120
TBE-Urea Buffer 2X 120
Components 4X Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40
  • Did not save images but could see digestion was incomplete (<1% completely undigested, ~50% half digested)
    • Increase USER incubation? Increase DpnII guide oligo concentration?

EtOH Precipitation[edit]

  • Put cut out gel in 8 0.5mL tubes (with holes at bottom) and centrifuged the tube at 15,000 rpm, for 3 min at RT.
  • Transferred the gel remaining in 0.5 mL tube to 2 mL tube below with pipette tip.
  • Added 450 ul of 1X TE buffer to each
  • Vortexed for 60min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transferred the clear spnt. to 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
  • Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transferred spnt to fresh 1.5 mL tube
  • Precipitated in 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 6 tubes at -80C overnight
  • Spun 8 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
  • Discard supernatant and let dry in hood for 10min
  • Resuspend each tube with 10ul and combine

Qubit Quantification[edit]

  • 10.3 ng/ul => 10.3 ng/ul / (157nt*325/nt + 79Da) = 201.5 nM (80ul)