Matt:LabNotes/2016-7-26: Difference between revisions

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===PAGE Size Selection===
===PAGE Size Selection===
*Run 10 gels
*Also include probes from [[Matt:LabNotes/2016-6-7]]
*200V for 40min
[[File:2016-07-28_TB12kApr2016_V4_ProbeProduction_GelCheck.jpg|350px]]
*You can see the 3 bands at the top
*The full digested band is the brightest (third from top) and also kind of blurry due to amount
 
*Run 6 gels for 6/7/2016 batch
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''6X Volume'''
|-
| V4 Probes||95
|-
| TBE-Urea Buffer 2X||95
|}


*Run 9 gels for this batch (wanted to do 10 but not enough gels)
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
| align="center" style="background:#f0f0f0;"|'''9X Volume'''
|-
|-
| V4 Probes||135
| V4 Probes||135
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{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
| align="center" style="background:#f0f0f0;"|'''15X Volume'''
|-
|-
| Low Mass Ladder||10
| Low Mass Ladder||15
|-
|-
| TBE-Urea Buffer 2X||50
| TBE-Urea Buffer 2X||75
|-
|-
| H2O||40
| H2O||60
|-
|-
| Total||100
| Total||150
|}
|}
*200V for 40min
*No gel images because too many gels
**Bands looked good, could see '''very''' faint band of ~200bp and a less faint band below that and finally the completely digested probe band was brightest below that


===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 30 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
*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 2 Nanosep columns and centrifuged at 15,000 rpm for 3 min
*Transferred the clear spnt. to 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 (~3500ul)
*Transferred spnt to fresh 1.5 mL tube
*Precipitated in 8 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
*Precipitated in 30 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
*Vortexed and placed the 8 tubes at -80C for 30min
*Vortexed and placed the 30 tubes at -80C for over the weekend
*Spun 8 tubes at 10,000rpm at 4C for 30min
*Spun 30 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===
20.2ng/ul x 74ul = 1,494.8ng
22.7ng/ul x 300ul = 6,810ng<br>
20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM-->
22.7ng/ul / (155nt x 303.7Da/nt + 79Da) = 478nM

Latest revision as of 19:01, 2 August 2016

TB12k_Apr2016 V4 Probe Preparation[edit]

Production PCR[edit]

  • Make 10ml V4 master mix for 96 wells
Components Volume (1X) Volume (100X)
First round amplicon TB12k_Apr2016_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 10,000

Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 4C hold

  • Did in regular thermocyclers

EtOH Precipitation[edit]

  • 8 15-ml tubes (with 12 wells (1.5 strips) of PCR product each) for
    • 1,200ul PCR product
    • 3000ul 100% EtOH
    • 4ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for 30 min
  • 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 8 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]

  • 8 columns elute 50ul each
  • Nanodrop: 374.4 ng/uL x 400uL = 149.76ug

Lambda Exo[edit]

  • Divide into 8 pcr tubes of 100ul (ideally should be <10ug each)
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 3hr (normally 1hr)
  • Purified with 8 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 167.0ng/ul x 320ul = 53.44ug

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 6 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 53.3
USER 5
10X DpnII Buffer 8
H2O 13.7
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 (100ul total)
  • Nanodrop
    • 229.0 ng/ul x 140ul = 32.06 ug (59% yield)

PAGE Size Selection[edit]

File:2016-07-28 TB12kApr2016 V4 ProbeProduction GelCheck.jpg

  • You can see the 3 bands at the top
  • The full digested band is the brightest (third from top) and also kind of blurry due to amount
  • Run 6 gels for 6/7/2016 batch
Components 6X Volume
V4 Probes 95
TBE-Urea Buffer 2X 95
  • Run 9 gels for this batch (wanted to do 10 but not enough gels)
Components 9X Volume
V4 Probes 135
TBE-Urea Buffer 2X 135
Components 15X Volume
Low Mass Ladder 15
TBE-Urea Buffer 2X 75
H2O 60
Total 150
  • 200V for 40min
  • No gel images because too many gels
    • Bands looked good, could see very faint band of ~200bp and a less faint band below that and finally the completely digested probe band was brightest below that

EtOH Precipitation[edit]

  • Put cut out gel in 30 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 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 30 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 30 tubes at -80C for over the weekend
  • Spun 30 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]

22.7ng/ul x 300ul = 6,810ng
22.7ng/ul / (155nt x 303.7Da/nt + 79Da) = 478nM