Matt:LabNotes/2015-6-3: Difference between revisions

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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
[[File:20150603_CA12kNov2014_V4_ProductionPCR.JPG|650px]]


===QIAEX-II Purification===
===QIAEX-II Purification===
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*Incubated at 37C for 1hr
*Incubated at 37C for 1hr20min
*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:
**ng/ul x 240ul = ug (76% yield)
**59.6ng/ul x 240ul = 14.3ug (61% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
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*Added 5ul DpnII
*Added 5ul DpnII
*Incubated at 37C for overnight (~15hrs)
*Incubated at 37C for overnight (~15hrs)
<!--
 
===Zymo Column Purification===
===Zymo Column Purification===
*Eluted 30ul each column (115ul total)
*Eluted 30ul each column (115ul total)
*Nanodrop
*Nanodrop
**81.2ng/ul x 115ul = 9.3ug (49% yield)
**51.9ng/ul x 115ul = 6.2ug (43% 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||115
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{| {{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
|}
|}
[[File:2015-06-05_CA12kNov2014_V4_SizeSelect.jpg]]


*Gel 1
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel1.jpg]]
*Gel 2
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_Gel2.jpg]]
*Gel 3
[[File:2015-02-04_CA12kNov14_SizeSelect_V4_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 4 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
*Vortexed for 120min 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 4 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 4 fresh 1.5 mL tube (~440ul per tube)
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
*Vortexed and placed the 2 tubes at -80C for 20min
*Vortexed and placed the 4 tubes at -80C for overnight
*Spun 6 tubes at 10,000rpm at 4C for 30min
*Spun 4 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
*Resuspend each tube with 10ul and combine
*Resuspend each tube with 10ul and combine


===TBU Gel Quantification===
===Qubit ssDNA Quantification===
*Dilute 2ul to 10ul with H2O
21.4ng/ul => 21.4 ng/ul / (150nt*325/nt + 79Da) = 438nM (60ul)
*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:2015-02-05_CA12kNov14_V4_GelQuant.jpg|650px]]
*Only use ladder bands 100bp, 200bp, to get trendline with better R^2
* 40ng/ul =>  40ng/ul / (150nt*325Da/nt + 79Da) = 819nM (58ul)
 
[[Media:2015-02-05_CA12kNov14_V4_GelQuant.xlsx | How I calculated concentration]]
-->

Latest revision as of 17:04, 7 June 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:20150603 CA12kNov2014 V4 ProductionPCR.JPG

QIAEX-II Purification[edit]

protocol

  • Divide into 8 5mL tubes of 1200ul each
  • Add 3600ul (3X volume) of Buffer QX1
  • Vortex the QIAEXII for 30sec
  • Add 30ul QIAEXII (30ul for every 10ug)
  • Incubate at 50C while mixing for 10min
    • Color should be yellow
  • Centrifuge sample for 30sec
  • Remove supernatant with a pipette
  • Wash the pellet with 500ul Buffer QX1
  • Resuspend the pellet by vortexing
  • Transfer to 1.5ml tubes
  • Centrifuge sample for 30sec and remove supernatant again with pipette
  • Wash the pellet twice with 500ul Buffer PE
  • Air-dry the pellet for 30min until the pellet turns white
  • To elute, resuspend with 20ul H2O and vortexing
  • Incubate at RT for 5min
  • Centrifuge for 30sec and pipette out supernatant with sample
  • Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
  • Total volume: 300ul
  • Nanodrop quantification: 155.7 ng/ul -> 46.7 ug

Lambda Exonuclease Digestion[edit]

  • Divide into 6 pcr tubes of 50ul with total amplicon of <10ug each
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 1hr20min
  • Purified with 6 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 59.6ng/ul x 240ul = 14.3ug (61% 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 30ul each column (115ul total)
  • Nanodrop
    • 51.9ng/ul x 115ul = 6.2ug (43% yield)

PAGE Size Selection[edit]

  • Run 2 gels
    • 200V for 40min
Components 2X Volume
V4 Probes 115
TBE-Urea Buffer 2X 115
Components 2X Volume
Low Mass Ladder 2
TBE-Urea Buffer 2X 10
H2O 8
Total 20

File:2015-06-05 CA12kNov2014 V4 SizeSelect.jpg

EtOH Precipitation[edit]

  • 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.
  • 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
  • Vortexed for >60min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transferred the clear spnt. to 4 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 4 fresh 1.5 mL tube (~440ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 4 tubes at -80C for overnight
  • Spun 4 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 ssDNA Quantification[edit]

21.4ng/ul => 21.4 ng/ul / (150nt*325/nt + 79Da) = 438nM (60ul)