Matt:LabNotes/2015-5-5: Difference between revisions

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>Mzcai
 
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*Purified with 6 Zymo ssDNA/RNA columns
*Purified with 6 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:<!--
*Recombined and measured ssDNA with Nanodrop:
**66.6 ng/ul x 240ul =  15.98 ug( 65% yield)
**50.4 ng/ul x 240ul =  12.1 ug (67% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
Line 106: Line 106:


===Zymo Column Purification===
===Zymo Column Purification===
*Eluted 30ul each column (115ul total)
*Eluted 20ul each column (80ul total)
*Nanodrop
*Nanodrop
**74.9ng/ul x 115ul = 8.6ug (54% yield)
**63.6ng/ul x 80ul = 5.0ug (41% yield)


===PAGE Size Selection===
===PAGE Size Selection===
*Run 3 gels
*Run 2 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;"|'''3X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| V4 Probes||115
| V4 Probes||80
|-
|-
| TBE-Urea Buffer 2X||115
| TBE-Urea Buffer 2X||80
|}
|}


{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''3X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| Low Mass Ladder||3
| Low Mass Ladder||2
|-
|-
| TBE-Urea Buffer 2X||15
| TBE-Urea Buffer 2X||10
|-
|-
| H2O||12
| H2O||8
|-
|-
| Total||30
| Total||20
|}
|}


*Gel 1
*The PCR tube (type with caps attached) used to heat denature sample had a leak
[[File:2015-03-03_CA12kNov14_V4_SizeSelect_Gel1.jpg]]
**~half of the sample leaked out so I only ran 1 gel
*Gel 2
[[File:2015-03-03_CA12kNov14_V4_SizeSelect_Gel2.jpg]]
*Gel 3
[[File:2015-03-03_CA12kNov14_V4_SizeSelect_Gel3.jpg]]


===EtOH Precipitation===
===EtOH Precipitation===
*Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Put cut out gel in 2 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 2.0 mL tube below with pipette tip.
*Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
*Added 900 ul of 1X TE buffer
*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
*Centrifuged at 15,000 rpm for 3 min at RT
*Centrifuged at 15,000 rpm for 3 min at RT
*Transferred the clear spnt. to 6 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 6 fresh 1.5 mL tube (~400ul per tube)
*Transferred spnt to fresh 1.5 mL tube (~850ul)
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Saved for EtOH precipitation on next batch of probe production
*Vortexed and placed the 2 tubes at -80C for overnight
*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 let dry in hood for 10min
*Resuspend each tube with 10ul and combine
 
===TBU Gel Quantification===
*Dilute 2ul to 10ul with H2O
*Add 10ul 2X TBE-Urea Buffer
*Load wells with 2, 4, 6, and 8 ul
*Load Low Mass DNA ladders in 0.5, 1, and 2 ul
*Ran at 200 V for 35min
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''0.5 ladder'''
| align="center" style="background:#f0f0f0;"|'''1 ladder'''
| align="center" style="background:#f0f0f0;"|''' 2 ul '''
| align="center" style="background:#f0f0f0;"|''' 4 ul '''
| align="center" style="background:#f0f0f0;"|''' 6 ul '''
| align="center" style="background:#f0f0f0;"|''' 8 ul '''
| align="center" style="background:#f0f0f0;"|'''2 ladder'''
|-
| DNA volume||0.5||1||0.1||0.2||0.3||0.4||2
|-
| H2O||1.5||1||0.9||1.8||2.7||3.6||0
|-
| 2X Buffer||2||2||1||2||3||4||2
|-
| Total||4||4||2||4||6||8||4
|-
|
|}
[[File:2015-03-04_CA12kNov14_V4_GelQuant.jpg|650px]]
*Only use ladder bands 100bp, 200bp, to get trendline with better R^2
*55.6 ng/ul => 55.6 ng/ul / (150nt*325Da/nt + 79Da) = 1.14uM (1,139nM) (58ul)
 
[[Media:2015-03-04_CA12kNov14_V4_GelQuant.xlsx | How I calculated concentration]]-->

Latest revision as of 22:35, 7 May 2015

CA12k_Nov2014_V4 Probe Production[edit]

Production PCR[edit]

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V4 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4900
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:20150505 CA12kNov2014 V4 ProductionPCR.JPG

EtOH Precipitation[edit]

  • 12 5-ml tubes (with 8 wells of PCR product each) for V4
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 30min
  • Centrifuged at 3000rpm at 4C for 30 min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 30 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification[edit]

  • Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~600ul of V4 probes: 59.8 ng/ul => ~35.9 ug

Lambda Exonuclease Digestion[edit]

  • Divide into 6 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 1hr
  • Purified with 6 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 50.4 ng/ul x 240ul = 12.1 ug (67% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 4 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 59
USER 5
10X DpnII Buffer 8
H2O 8
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
    • 63.6ng/ul x 80ul = 5.0ug (41% yield)

PAGE Size Selection[edit]

  • Run 2 gels
    • 200V for 40min
Components 2X Volume
V4 Probes 80
TBE-Urea Buffer 2X 80
Components 2X Volume
Low Mass Ladder 2
TBE-Urea Buffer 2X 10
H2O 8
Total 20
  • The PCR tube (type with caps attached) used to heat denature sample had a leak
    • ~half of the sample leaked out so I only ran 1 gel

EtOH Precipitation[edit]

  • Put cut out gel in 2 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.
  • 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 (~850ul)
  • Saved for EtOH precipitation on next batch of probe production