Matt:LabNotes/2016-6-7: Difference between revisions

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*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**118.7ng/ul x 280ul = 33.24ug
**118.7ng/ul x 280ul = 33.24ug
<!--
 
===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====
Line 74: Line 74:
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| SS-amplicon||64
| SS-amplicon||56
|-
|-
| USER||5
| USER||5
Line 80: Line 80:
| 10X DpnII Buffer||8
| 10X DpnII Buffer||8
|-
|-
| H2O||3
| H2O||11
|-
|-
| Total||80
| Total||80
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*Eluted 20ul each column (100ul total)
*Eluted 20ul each column (100ul total)
*Nanodrop
*Nanodrop
**147.2 ng/ul x 100ul = 14.7ug (50.4% yield)
**193.7 ng/ul x 100ul = 19.37 ug (% yield)
 
===PAGE Size Selection===
*Run 4 gels
*200V for 40min
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
|-
| V4 Probes||95
|-
| TBE-Urea Buffer 2X||95
|}
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''4X Volume'''
|-
| Low Mass Ladder||4
|-
| TBE-Urea Buffer 2X||20
|-
| H2O||16
|-
| Total||40
|}
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel1.jpg|350px]]<br>
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel2.jpg|350px]]<br>
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel3.jpg|350px]]<br>
[[File:2016-06-02_TB12kApr2016_V4_SizeSelect_Gel4.jpg|350px]]<br>
 
===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.
*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 (~3500ul)
*Precipitated in 8 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
*Spun 8 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===
===Continued on [[Matt:LabNotes/2016-7-26]]===
20.2ng/ul x 74ul = 1,494.8ng
20.2ng/ul / (157nt x 303.7Da/nt + 79Da) = 423nM-->

Latest revision as of 19:39, 1 August 2016

TB12k_Apr2016 V4 Probe Preparation[edit]

Production PCR[edit]

  • Make 2X V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon TB12k_Apr2016_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

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

  • No curve because 100ul reaction volume only works in Biorad PCR machines

EtOH Precipitation[edit]

  • 8 15-ml tubes (with 24 wells (3 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 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 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: 313.6 ng/uL x 400uL = 125.44ug

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 2hr (normally 1hr)
  • Purified with 8 Zymo ssDNA/RNA columns
    • Dropped one column so only 7
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 118.7ng/ul x 280ul = 33.24ug

Remove Amplification Adapters[edit]

USER[edit]

  • Split into 5 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 56
USER 5
10X DpnII Buffer 8
H2O 11
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
    • 193.7 ng/ul x 100ul = 19.37 ug (% yield)

Continued on Matt:LabNotes/2016-7-26[edit]