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

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**V7: 50ul x 142nM = '''710ul''' x 10nM
**V7: 50ul x 142nM = '''710ul''' x 10nM
***Add 660ul H2O
***Add 660ul H2O
====Gel Check====
*Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
*Use 10nM Agi26k0gap expansion PCR'd probes as positive control
*Although band is '''very''' faint it can be seen at slightly under 200bp
[[File:2015-01-06_1stRndAmplicon_V4V7_Gel.jpg]]


===Production PCR===
===Production PCR===
Line 95: Line 101:
*Discarded supernatant and added 800ul of cold 80% EtOH
*Discarded supernatant and added 800ul of cold 80% EtOH
*Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
*Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
*Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
*Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
*Discarded supernatant and air-dried for 5 min in hood
*Discarded supernatant and air-dried for 5 min in hood
*Resuspended DNA with 100ul H2O
*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
{| {{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 4 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
**66.5ng/ul x 160ul = 10.6ug (57.3% yield)
**56.9ng/ul x 160ul = 9.1ug (55.2% yield)
===Remove Amplification Adapters===
====USER====
*Split each set into 2 PCR tubes and add 5ul USER
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| SS-amplicon||80
|-
| USER||5
|-
| Total||85
|}
*Incubate at 37C for 2.5 hours
====DpnII====
*Added 15ul of the following
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X DpnII Buffer||10
|-
| 100uM RE-DpnII guide oligo||5
|}
  RE-DpnII guide oligo:
  V4: RE DpnII V4
  V7: RE DpnII V7
*Incubated at 94C for 2 min, then 37C for 3 min
*Added 5ul DpnII
*Incubated at 37C for 4 hr
===TBU Gel: Digestion Check===
*Combine into 1 tube for each set
*2ul Digested Sample + 2ul 2X Urea Loading Dye
*5ul Undigested sample (10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
[[File:2015-01-07_V4V7_Digest_GelCheck.jpg]]
*Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters
*To get a higher yield I continued digestion overnight
===Continue Digestion===
*Add 100ul of the following to each tube
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| 10X DpnII Buffer||10
|-
| USER||5
|-
| H2O||85
|-
| Total||100
|}
*Incubated at 37C for overnight (15 hrs)
===Zymo Column Purification===
*4 columns for each set
*Eluted 30ul each column (120ul total)
*Nanodrop
**V4: 45.0ng/ul x 120ul = 5.4ug
**V7: 22.8ng/ul x 120ul = 2.7ug (oddly low but checked with nanodrop twice)
===PAGE Size Selection===
*Run 2 gels for V4 and 1 gel for V7
**220V for 30min
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
| V4 Probes||120
|-
| TBE-Urea Buffer 2X||120
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
|-
| V7 Probes||120
|-
| TBE-Urea Buffer 2X||120
|}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| Low Mass Ladder||3
|-
| TBE-Urea Buffer 2X||15
|-
| H2O||12
|-
| Total||30
|}
*V4 Gel 1
[[File:2015-01-08_SizeSelect_V4_Gel1.jpg]]
*V4 Gel 2
[[File:2015-01-08_SizeSelect_V4_Gel2.jpg]]
*V7 Gel 1
**Had trouble seeing blade and may have cut some extra above where I should have. However a mitigating factor is that V7 was digested much more completely than V4 based on intensity of band before cutting
[[File:2015-01-08_SizeSelect_V7_Gel1.jpg]]
===EtOH Precipitation===
*Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
*Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
*Added 900 ul of 1X TE buffer
*Vortexed for 60 min at 37 C in incubator
*Centrifuged at 15,000 rpm for 3 min at RT
*Transfered the clear spnt. to 6 Nanosep column and centrifuged at 15,000 rpm for 3 min
*Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
*Transfered spnt to fresh 1.5 mL tube (~400ul per tube)
*Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
*Vortexed and placed the 6 tubes at -80C for overnight
*Spun 6 tubes at 10,000rpm at 4C for 30min
*Discard supernatant and add 650ul 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 recombine (40ul for V4 and 20ul for V7)
===TBU Gel Quantification===
*Dilute 2ul to 10ul with H2O
*Add 10ul 2X TBE-Urea Buffer
*Load wells with 2, 4, 6, and 8 ul
*Load Low Mass DNA ladders in 0.5, 1, and 2 ul
*Ran at 200 V for 35min
{| {{table}}
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''0.5 ladder'''
| align="center" style="background:#f0f0f0;"|'''1 ladder'''
| align="center" style="background:#f0f0f0;"|''' 2 ul '''
| align="center" style="background:#f0f0f0;"|''' 4 ul '''
| align="center" style="background:#f0f0f0;"|''' 6 ul '''
| align="center" style="background:#f0f0f0;"|''' 8 ul '''
| align="center" style="background:#f0f0f0;"|'''2 ladder'''
|-
| DNA volume||0.5||1||0.1||0.2||0.3||0.4||2
|-
| H2O||1.5||1||0.9||1.8||2.7||3.6||0
|-
| 2X Buffer||2||2||1||2||3||4||2
|-
| Total||4||4||2||4||6||8||4
|-
|
|}
[[File:2015-01-09_V4_GelQuant.jpg|650px]]
[[File:2015-01-09_V7_GelQuant.jpg|650px]]
*Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear
*V4: 10.7 ng/ul => 10.7 ng/ul / (150nt*325Da/nt + 79Da) = 219nM (38ul)
*V7: 6.2 ng/ul => 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul)
[[Media:2015-01-09_V4V7_GelQuant.xlsx | How I calculated concentration]]

Latest revision as of 22:33, 12 January 2015

CA12k_Nov2014 Probe Production[edit]

Expansion PCR[edit]

  • 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

Gel Check[edit]

  • Use 5ul of 10nM expansion PCR'd probes + 5ul 2X Loading dye
  • Use 10nM Agi26k0gap expansion PCR'd probes as positive control
  • Although band is very faint it can be seen at slightly under 200bp

File:2015-01-06 1stRndAmplicon V4V7 Gel.jpg

Production PCR[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • 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 4 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 66.5ng/ul x 160ul = 10.6ug (57.3% yield)
    • 56.9ng/ul x 160ul = 9.1ug (55.2% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split each set into 2 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 80
USER 5
Total 85
  • Incubate at 37C for 2.5 hours

DpnII[edit]

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 10
100uM RE-DpnII guide oligo 5
 RE-DpnII guide oligo:
 V4: RE DpnII V4
 V7: RE DpnII V7
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for 4 hr

TBU Gel: Digestion Check[edit]

  • Combine into 1 tube for each set
  • 2ul Digested Sample + 2ul 2X Urea Loading Dye
  • 5ul Undigested sample (10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
  • 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye

File:2015-01-07 V4V7 Digest GelCheck.jpg

  • Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters
  • To get a higher yield I continued digestion overnight

Continue Digestion[edit]

  • Add 100ul of the following to each tube
Components Volume
10X DpnII Buffer 10
USER 5
H2O 85
Total 100
  • Incubated at 37C for overnight (15 hrs)

Zymo Column Purification[edit]

  • 4 columns for each set
  • Eluted 30ul each column (120ul total)
  • Nanodrop
    • V4: 45.0ng/ul x 120ul = 5.4ug
    • V7: 22.8ng/ul x 120ul = 2.7ug (oddly low but checked with nanodrop twice)

PAGE Size Selection[edit]

  • Run 2 gels for V4 and 1 gel for V7
    • 220V for 30min
Components 2X Volume
V4 Probes 120
TBE-Urea Buffer 2X 120
Components 1X Volume
V7 Probes 120
TBE-Urea Buffer 2X 120
Components Volume
Low Mass Ladder 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • V4 Gel 1

File:2015-01-08 SizeSelect V4 Gel1.jpg

  • V4 Gel 2

File:2015-01-08 SizeSelect V4 Gel2.jpg

  • V7 Gel 1
    • Had trouble seeing blade and may have cut some extra above where I should have. However a mitigating factor is that V7 was digested much more completely than V4 based on intensity of band before cutting

File:2015-01-08 SizeSelect V7 Gel1.jpg

EtOH Precipitation[edit]

  • Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.
  • Transfered the gel remaining in 0.5 mL tube to 2.0 mL tube below with pipette tip.
  • Added 900 ul of 1X TE buffer
  • Vortexed for 60 min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to 6 Nanosep column and centrifuged at 15,000 rpm for 3 min
  • Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
  • Transfered spnt to fresh 1.5 mL tube (~400ul per tube)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
  • Vortexed and placed the 6 tubes at -80C for overnight
  • Spun 6 tubes at 10,000rpm at 4C for 30min
  • Discard supernatant and add 650ul 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 recombine (40ul for V4 and 20ul for V7)

TBU Gel Quantification[edit]

  • Dilute 2ul to 10ul with H2O
  • Add 10ul 2X TBE-Urea Buffer
  • Load wells with 2, 4, 6, and 8 ul
  • Load Low Mass DNA ladders in 0.5, 1, and 2 ul
  • Ran at 200 V for 35min
' 0.5 ladder 1 ladder 2 ul 4 ul 6 ul 8 ul 2 ladder
DNA volume 0.5 1 0.1 0.2 0.3 0.4 2
H2O 1.5 1 0.9 1.8 2.7 3.6 0
2X Buffer 2 2 1 2 3 4 2
Total 4 4 2 4 6 8 4

File:2015-01-09 V4 GelQuant.jpg File:2015-01-09 V7 GelQuant.jpg

  • Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear
  • V4: 10.7 ng/ul => 10.7 ng/ul / (150nt*325Da/nt + 79Da) = 219nM (38ul)
  • V7: 6.2 ng/ul => 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul)

How I calculated concentration