Matt:LabNotes/2016-9-14: Difference between revisions

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>Mzcai
(Created page with "==CA12k_Nov2014 V6 (RevComp of V4) Probe Production== * Expansion PCR and first time Production PCR ===Production PCR=== *V6 master mix {| {{tab...")
 
>Mzcai
 
(10 intermediate revisions by the same user not shown)
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| Total||100||5000
| Total||100||5000
|}
|}
<!--
 
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
*Stopped after 15 cycles
[[File:012714_CA12kNov14_ProductionPCR_V6V8.JPG | 800px]]


===EtOH Precipitation===
===EtOH Precipitation===
*6 5-ml tubes (with 8 wells of PCR product each) for V6 and 6 tubes for V8
*12 5-ml tubes (with 8 wells of PCR product each)
**800ul PCR product
**800ul PCR product
**2000ul 100% EtOH
**2000ul 100% EtOH
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*Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
*Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:
 
~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug
~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide each into 6 pcr tubes of 48ul with total amplicon of <10ug each
*Divide each into 8 pcr tubes of 75ul with total amplicon of <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||48
| Amplicon||75
|-
|-
| 10X Lambda Exo Buffer||10
| 10X Lambda Exo Buffer||10
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| Lambda Exonuclease||10
| Lambda Exonuclease||10
|-
|-
| H2O||32
| H2O||5
|-
|-
| Total||100
| Total||100
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*Incubated at 37C for 1hr
*Incubated at 37C for 1hr
*Purified with 6 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:
**V6: 64.0ng/ul x 240ul = 15.4ug (67% yield)
**V6: 80.3ng/ul x 320ul = 25.7ug (75.6% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====
*Split each set into 3 PCR tubes and add 5ul USER
*Split each set into 5 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||80
| SS-amplicon||63
|-
|-
| USER||5
| USER||5
|-
|-
| Total||85
| DpnII 10X||8
|-
| H2O||4
|-
| Total||80
|}
|}
*Incubate at 37C for 3 hours
*Incubate at 37C for 3 hours
====DpnII====
====DpnII====
*Added 15ul of the following
*Added 15ul of the following
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| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| 10X DpnII Buffer||10
| 10X DpnII Buffer||2
|-
| 100uM RE-DpnII_V6 guide oligo||5
|-
| H2O||8
|-
|-
| 100uM RE-DpnII guide oligo||5
|}
|}
  RE-DpnII guide oligo:
 
  V6: RE DpnII V6
*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 (16 hrs)
*Incubated at 37C for overnight (12 hrs)
 
===TBU Gel: Digestion Check===
*Combine into 1 tube for each set
*2ul Digested Sample + 2ul 2X Urea Loading Dye
*5ul Undigested sample (10nM V8 1st round amplicon) + 5ul 2X Urea Loading Dye
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
[[File:]]


===Zymo Column Purification===
===Zymo Column Purification===
*3 columns for each set
*Eluted 20ul each column (100ul total)
*Eluted 20ul each column (60ul total)
*Nanodrop
*Nanodrop
**V6: 137.3ng/ul x 60ul = 8.2ug
**V6: 118.9ng/ul x 100ul = 11.9ug


===PAGE Size Selection===
===PAGE Size Selection===
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{| {{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'''
|-
|-
| V6 Probes||60
| V6 Probes||100
|-
|-
| H2O ||60
| H2O||20
|-
|-
| TBE-Urea Buffer 2X||120
| TBE-Urea Buffer 2X||120
Line 142: Line 138:


===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 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.)
*Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
*Transfered 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 60 min at 37 C in incubator
*Vortexed for 60 min 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
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*Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min
*Discard supernatant and add 650ul 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 recombine (40ul total each)
*Resuspend each tube with 10ul and recombine (80ul total)
 
*Measure with qubit
[[File:2015-01-30_V6_GelQuant.jpg|650px]]
*V6: 13.1 ng/ul => 13.1 ng/ul / (150nt*325Da/nt + 79Da) = 268.2nM (80ul)
*V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul)
 
[[Media:2015-01-30_V6V7_GelQuant.xlsx | How I calculated concentration]]
-->

Latest revision as of 22:20, 21 September 2016

CA12k_Nov2014 V6 (RevComp of V4) Probe Production[edit]

Production PCR[edit]

  • V6 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V6 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V6U (100uM) 0.4 20
AP2V6 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold

EtOH Precipitation[edit]

  • 12 5-ml tubes (with 8 wells of PCR product each)
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 1hr
  • 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 5 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 V6 probes into one 1.5ml and all V8 probes into another
  • Measured concentration with Nanodrop:

~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug

Lambda Exonuclease Digestion[edit]

  • Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each
Components Volume
Amplicon 75
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 5
Total 100
  • Incubated at 37C for 1hr
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • V6: 80.3ng/ul x 320ul = 25.7ug (75.6% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split each set into 5 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 63
USER 5
DpnII 10X 8
H2O 4
Total 80
  • Incubate at 37C for 3 hours

DpnII[edit]

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 2
100uM RE-DpnII_V6 guide oligo 5
H2O 8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (12 hrs)

Zymo Column Purification[edit]

  • Eluted 20ul each column (100ul total)
  • Nanodrop
    • V6: 118.9ng/ul x 100ul = 11.9ug

PAGE Size Selection[edit]

  • Run 4 gels for V6
    • 200V for 35min
Components 4X Volume
V6 Probes 100
H2O 20
TBE-Urea Buffer 2X 120
Components Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40

EtOH Precipitation[edit]

  • 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.)
  • Transfered 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 60 min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to 8 Nanosep column and centrifuged at 15,000 rpm for 3 min
  • Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transfered spnt to fresh 1.5 mL tube (~400ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 8 tubes at -80C for 30 min
  • Spun 8 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul 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 recombine (80ul total)
  • Measure with qubit
  • V6: 13.1 ng/ul => 13.1 ng/ul / (150nt*325Da/nt + 79Da) = 268.2nM (80ul)