Matt:LabNotes/2017-6-6: Difference between revisions

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>Mzcai
(Created page with "==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8 ===Production PCR=== *Matt:LabNotes/2017-3-30 | 25nM 1st round amplicon...")
 
>Mzcai
 
(5 intermediate revisions by the same user not shown)
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*Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
*Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
*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: 244.2 ng/ul => ~97.64 ug
~400ul of V4 probes: 248.3 ng/ul => ~99.3 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
Line 61: Line 61:


*Incubate 4 tubes at 37C for 2hr
*Incubate 4 tubes at 37C for 2hr
*Purified with 8 Zymo ssDNA/RNA columns
*Purified with 7 Zymo ssDNA/RNA columns (one tube turned solution yellow in IIC column)
*Eluted with 40ul each
*Eluted with 46ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**2hr incubation: 81.7 ng/ul x 160ul 13 ug
**2hr incubation: 88.1 ng/ul x 320ul 28 ug


===Remove Amplification Adapters===
===Remove Amplification Adapters===
Line 106: Line 106:
*Eluted 20ul each column (80ul total)
*Eluted 20ul each column (80ul total)
*Nanodrop
*Nanodrop
**125.1ng/ul x 120ul = 15ug (% yield)
**150.1ng/ul x 120ul = 18ug (% yield)


===PAGE Size Selection===
===PAGE Size Selection===
Line 135: Line 135:
===EtOH Precipitation===
===EtOH Precipitation===
*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.
*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 2.0 mL tube below with pipette tip.
**Had to use 2 more 1.5mL tubes to get all gel. Use 2.0mL tubes next time
*Added 450 ul of 1X TE buffer to each
*Added 450 ul of 1X TE buffer to each
*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 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
*Transferred spnt to fresh 1.5 mL tube
*Precipitated in 10 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
*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 10 tubes at -80C overnight
*Vortexed and placed the 8 tubes at -80C 30min
*Spun 10 tubes at 10,000rpm at 4C for 30min
*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 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 8ul and combine
*Resuspend each tube with 10ul and combine


===Qubit Quantification===
===Qubit Quantification===
*16.7 ng/ul => 16.7 ng/ul / (157nt*325/nt + 79Da) = 326.7 nM (80ul)
*22.9 ng/ul => 22.9 ng/ul / (157nt*325/nt + 79Da) = 448.1 nM (80ul)
-->

Latest revision as of 00:07, 18 June 2017

Agi15k_Feb2017_V4 Probe Production[edit]

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 13 -> 72C 2min -> 15C hold

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 3,000rpm 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 5 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: 248.3 ng/ul => ~99.3 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
  • Incubate 4 tubes at 37C for 2hr
  • Purified with 7 Zymo ssDNA/RNA columns (one tube turned solution yellow in IIC column)
  • Eluted with 46ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 2hr incubation: 88.1 ng/ul x 320ul = 28 ug

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
    • 150.1ng/ul x 120ul = 18ug (% 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

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 2.0 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 8 tubes at -80C 30min
  • 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]

  • 22.9 ng/ul => 22.9 ng/ul / (157nt*325/nt + 79Da) = 448.1 nM (80ul)