Matt:LabNotes/2016-11-10: Difference between revisions

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===Qia Column Purification===
===Qia Column Purification===
*8 columns elute 50ul each<!--
*16 columns elute 50ul each
*Nanodrop: 208.8 ng/uL x 200uL = 41.76ug
*Nanodrop: 170.3 ng/uL x 800uL = 136.24ug


===Lambda Exo===
===Lambda Exo===
*Divide into 4 pcr tubes of 100ul (ideally should be <10ug each)
*Divide into 14 pcr tubes of 100ul (ideally should be <10ug each)
{| {{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'''
|-
|-
| Amplicon||50
| Amplicon||57
|-
|-
| 10X Lambda Exo Buffer||10
| 10X Lambda Exo Buffer||10
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| Lambda Exonuclease||10
| Lambda Exonuclease||10
|-
|-
| H2O||30
| H2O||23
|-
|-
| Total||100
| Total||100
Line 61: Line 61:


*Incubated at 37C for 1hr
*Incubated at 37C for 1hr
*Purified with 4 Zymo ssDNA/RNA columns
*Purified with 14 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**96.8ng/ul x 160ul = 15.49ug (74% yield)
**72.4ng/ul x 560ul = 40.54ug (60% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
*Save 4ul for gel check of size
====USER====
====USER====
*Split into 3 PCR tubes and add 5ul USER
*Split into 9 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||52
| SS-amplicon||62.2
|-
|-
| USER||5
| USER||5
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| 10X DpnII Buffer||8
| 10X DpnII Buffer||8
|-
|-
| H2O||15
| H2O||4.8
|-
|-
| Total||80
| Total||80
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*Incubated at 94C for 2 min, then 37C for 3 min
*Incubated at 94C for 2 min, then 37C for 3 min
*Added 5ul DpnII
*Added 5ul DpnII
*Incubated at 37C for overnight (~15hrs)
*Incubated at 37C for overnight (~12hrs)


===Zymo Column Purification===
===Zymo Column Purification===
*Eluted 20ul each column (60ul total)
*Eluted 20ul each column (180ul total)
*Nanodrop
*Nanodrop
**87.3 ng/ul x 60ul = 5.2ug (34% yield lower than normal)
**80.1 ng/ul x 180ul = 14.4ug (36% yield lower than normal)
 
===Gel Check===
*Pre-run 12 well TBU gel for 20min at 250V
*Rinse wells
#Ladder
#*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X TBU Loading Dye
#User/DpnII-Digested
#*2ul Sample + 2ul 2X TBU Loading Dye
#Lambda-Digested
#*2ul Sample + 2ul 2X TBU Loading Dye
*Pre-heat samples to 75C for 10min and then put on ice
*Load samples in middle lanes of gel
*Run for 33min at 210V
[[File:2016-11-08_CA12kOct2016_V4_GelCheck.jpg|250px]]


===PAGE Size Selection===
===PAGE Size Selection===
*Run 2 gels
*Run 3 gels (normally should be 5 gels to keep < 3ug per gel)
*200V for 40min
*200V for 40min


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| align="center" style="background:#f0f0f0;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| V4 Probes||60
| V4 Probes||180
|-
|-
| TBE-Urea Buffer 2X||60
| TBE-Urea Buffer 2X||180
|}
|}


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| align="center" style="background:#f0f0f0;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| Low Mass Ladder||2
| Low Mass Ladder||3
|-
|-
| TBE-Urea Buffer 2X||10
| TBE-Urea Buffer 2X||15
|-
|-
| H2O||8
| H2O||12
|-
|-
| Total||20
| Total||30
|}
|}
[[File:|350px]]<br>
[[File:|350px]]<br>


===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 6 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 1.5 mL tube below with pipette tip.
*Added 450 ul of 1X TE buffer to each
*Added 450 ul of 1X TE buffer to each
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*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
*Transferred spnt to fresh 1.5 mL tube
*Precipitated in 4 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
*Precipitated in 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
*Vortexed and placed the 4 tubes at -80C for 30min
*Vortexed and placed the 6 tubes at -80C for 30min
*Spun 4 tubes at 10,000rpm at 4C for 30min
*Spun 6 tubes at 10,000rpm at 4C for 30min
*Discard supernatant and add 750ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
*Discard supernatant and add 750ul 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
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===Qubit ssDNA===
===Qubit ssDNA===
990pg/ul x 40ul = 39.6ng
7.79ng/ul x 60ul = 467.4ng
990pg/ul / (157 x 303.7Da/nt + 79Da) = 20.7nM
7.79ng/ul / (157 x 303.7Da/nt + 79Da) = 160nM
-->

Latest revision as of 23:49, 15 November 2016

CA12k_Oct2016 V4 Probe Preparation[edit]

Production PCR[edit]

  • V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Oct2016_V4 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V4U (100uM) 0.4 20
AP2V4 (100uM) 0.4 20
H2O 49 2450
Total 100 5000
  • Did one plate - 96 wells 100ul each

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold 450px

EtOH Precipitation[edit]

  • 16 5-ml tubes each with
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • 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 16 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification[edit]

  • 16 columns elute 50ul each
  • Nanodrop: 170.3 ng/uL x 800uL = 136.24ug

Lambda Exo[edit]

  • Divide into 14 pcr tubes of 100ul (ideally should be <10ug each)
Components Volume
Amplicon 57
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 23
Total 100
  • Incubated at 37C for 1hr
  • Purified with 14 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 72.4ng/ul x 560ul = 40.54ug (60% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 9 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 62.2
USER 5
10X DpnII Buffer 8
H2O 4.8
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 (~12hrs)

Zymo Column Purification[edit]

  • Eluted 20ul each column (180ul total)
  • Nanodrop
    • 80.1 ng/ul x 180ul = 14.4ug (36% yield lower than normal)

PAGE Size Selection[edit]

  • Run 3 gels (normally should be 5 gels to keep < 3ug per gel)
  • 200V for 40min
Components 2X Volume
V4 Probes 180
TBE-Urea Buffer 2X 180
Components 2X Volume
Low Mass Ladder 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30

EtOH Precipitation[edit]

  • Put cut out gel in 6 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
  • Precipitated in 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 6 tubes at -80C for 30min
  • Spun 6 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 750ul 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[edit]

7.79ng/ul x 60ul = 467.4ng 7.79ng/ul / (157 x 303.7Da/nt + 79Da) = 160nM