Matt:LabNotes/2015-1-26: Difference between revisions

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*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:


~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug
~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug <br>
~290ul of V8 probes: 183.1 ng/ul => ~ 53 ug
~290ul of V8 probes: 183.1 ng/ul => ~ 53 ug
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===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide each into 4 pcr tubes of 72.5ul with total amplicon of <10ug each
*Divide each into 5 pcr tubes of 58ul 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||72.5
| Amplicon||58
|-
|-
| 10X Lambda Exo Buffer||10
| 10X Lambda Exo Buffer||10
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| Lambda Exonuclease||10
| Lambda Exonuclease||10
|-
|-
| H2O||7.5
| H2O||22
|-
|-
| Total||100
| Total||100
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*Incubated at 37C for 1hr
*Incubated at 37C for 1hr
*Purified with 4 Zymo ssDNA/RNA columns
*Purified with 5 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
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====USER====
====USER====
*Split each set into 2 PCR tubes and add 5ul USER
*Split each set into 2 PCR tubes and add 5ul USER
*Save 4ul of each for TBU gel check later
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''

Revision as of 17:03, 28 January 2015

CA12k_Nov2014 RevComp Probe Production

Expansion PCR

  • Matt:LabNotes/2014-12-18 conclusion: 21 cycles of PCR starting with 100nM seed oligos
  • Make 2 tubes:
    • V6 primers - CA12k_Nov2014_Contig_RevComp
    • V8 primers - CA12k_Nov2014_Const_RevComp
' Contig of Exons RevComp(V6) Constitutive Exons RevComp(V8)
CA12k_Nov2014 Seed Oligos (900nM) 15 15
2X KAPA SYBG MM 67.5 67.5
100uM AP1V6U 0.6 0
100uM AP2V6 0.6 0
100uM AP1V8U 0 0.6
100uM AP2V8 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:012614 CA12kNov14 ExpansionPCR V6V8.JPG

  • Did not think V6 amplified correctly (possibly due to underpipetting a primer) but V4 showed a similar curve
    • Nanodrop also shows V6 amplified as much or more than V4
  • Purified each with 1 Qiagen column and eluted with 50ul H2O
  • Nanodrop:
    • V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM
    • V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM
  • Diluted to 10nM
    • V6: 48ul x 95nM = 456ul x 10nM
      • Add 408ul H2O
    • V8: 48ul x 129nM = 619ul x 10nM
      • Add 571ul H2O

Gel Check

  • Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
  • Use 2ul 10nM Agi26k0gap expansion PCR'd probes as positive control

File:2015-01-26 1stRndAmplicon V6V8 Gel.jpg

  • Apparently Nanodrop is not accurate enough at low conc
  • V6 is missing a band at 188bp...
    • Try Expansion PCR with V6 primers on the last of the seed oligos

V6 Expansion PCR Try 2

Components Volume
CA12k_Nov2014 Seed Oligos (900nM) 14
2X KAPA SYBG MM 50
100uM AP1V6U 1
100uM AP2V6 1
H2O 34
Total 100

Program 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 30 -> 72C 2min -> 15C hold File:012614 CA12kNov14 ExpansionPCR V6 try2.JPG

  • Stopped after 22 cycles
  • Purified each with 1 Qiagen column and eluted with 50ul H2O
  • Nanodrop:
    • V6: 17.9 ng/ul -> 17.9 ng/ul / (188bp*660Da/bp) = 144nM
  • Diluted to 10nM
    • V6: 48ul x 144nM = 691ul x 10nM
      • Add 643ul H2O

Production PCR

  • 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
  • V8 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V8 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V8U (100uM) 0.4 20
AP2V8 (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:012714 CA12kNov14 ProductionPCR V6V8.JPG

EtOH Precipitation

  • 6 5-ml tubes (with 8 wells of PCR product each) for V6 and 6 tubes for V8
    • 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

  • 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:

~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug
~290ul of V8 probes: 183.1 ng/ul => ~ 53 ug

Lambda Exonuclease Digestion

  • Divide each into 5 pcr tubes of 58ul with total amplicon of <10ug each
Components Volume
Amplicon 58
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 22
Total 100
  • Incubated at 37C for 1hr
  • Purified with 5 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • ng/ul x ul = ug (% yield)
    • ng/ul x ul = ug (% yield)