Matt:LabNotes/2015-1-5: Difference between revisions

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~290ul of V4 probes: 127.8 ng/ul => ~37 ug
~290ul of V4 probes: 127.8 ng/ul => ~37 ug
~290ul of V7 probes: 113.9 ng/ul => ~33 ug
~290ul of V7 probes: 113.9 ng/ul => ~33 ug
===Lambda Exonuclease Digestion===
*Divide each into 4 pcr tubes of 72.5ul with total amplicon of <10ug each
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| Amplicon||72.5
|-
| 10X Lambda Exo Buffer||10
|-
| Lambda Exonuclease||10
|-
| H2O||7.5
|-
| Total||100
|}
*Incubated at 37C for 1hr
*Purified with Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
** ng/ul x ul = ug (63.6% yield)

Revision as of 22:56, 6 January 2015

CA12k_Nov2014 Probe Production

Expansion PCR

  • Matt:LabNotes/2014-12-18 conclusion: 21 cycles of PCR starting with 100nM seed oligos
  • Make 2 tubes:
    • V4 primers - CA12k_Nov2014_Contig
    • V7 primers - CA12k_Nov2014_Const
' Contig of Exons (V4) Constitutive Exons (V7)
CA12k_Nov2014 Seed Oligos (900nM) 15 15
2X KAPA SYBG MM 67.5 67.5
100uM AP1V4U 0.6 0
100uM AP2V4 0.6 0
100uM AP1V7U 0 0.6
100uM AP2V7 0 0.6
H2O 51.3 51.3
Total 135 135

Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold

File:010514 CA12kNov14 ExpansionPCR V4V7.JPG

  • Purified each with 1 Qiagen column and eluted with 50ul H2O
  • Nanodrop:
    • V4: 10.8 ng/ul -> 10.8 ng/ul / (188bp*660Da/bp) = 87nM
    • V7: 17.8 ng/ul -> 17.8 ng/ul / (190bp*660Da/bp) = 142nM
  • Diluted to 10nM
    • V4: 50ul x 87nM = 435ul x 10nM
      • Add 385ul H2O
    • V7: 50ul x 142nM = 710ul x 10nM
      • Add 660ul H2O

Production PCR

  • V4 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_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
  • V7 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V7 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V7U (100uM) 0.4 20
AP2V7 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold

  • Stopped after 15 cycles

File:010514 CA12kNov14 ProductionPCR V4V7.JPG

EtOH Precipitation

  • 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 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 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

  • Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another
  • Measured concentration with Nanodrop:

~290ul of V4 probes: 127.8 ng/ul => ~37 ug ~290ul of V7 probes: 113.9 ng/ul => ~33 ug

Lambda Exonuclease Digestion

  • Divide each into 4 pcr tubes of 72.5ul with total amplicon of <10ug each
Components Volume
Amplicon 72.5
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 7.5
Total 100
  • Incubated at 37C for 1hr
  • Purified with Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • ng/ul x ul = ug (63.6% yield)