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

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>Mzcai
(Created page with "==CA12k_Nov2014 RevComp Probe Production== *Production of Padlock Probes targeting cDNA (V6 and V8 primers) of Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering...")
 
>Mzcai
 
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'''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold
'''Program''' 95C 30sec -> (95C 5sec -> 55C 30sec-> 72C 45sec) x 21 -> 72C 2min -> 15C hold
<!--
 
[[File:010514_CA12kNov14_ExpansionPCR_V4V7.JPG]]
[[File:012614_CA12kNov14_ExpansionPCR_V6V8.JPG|650px]]
*Did not think V6 amplified correctly (possibly due to underpipetting a primer) but [[Matt:LabNotes/2015-1-5#Expansion_PCR|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
*Purified each with 1 Qiagen column and eluted with 50ul H2O
*Nanodrop:
*Nanodrop:
**V4: 10.8 ng/ul -> 10.8 ng/ul / (188bp*660Da/bp) = 87nM
**V6: 11.8 ng/ul -> 11.8 ng/ul / (188bp*660Da/bp) = 95nM
**V7: 17.8 ng/ul -> 17.8 ng/ul / (190bp*660Da/bp) = 142nM
**V8: 16.2 ng/ul -> 16.2 ng/ul / (190bp*660Da/bp) = 129nM


*Diluted to 10nM
*Diluted to 10nM
**V4: 50ul x 87nM = '''435ul''' x 10nM
**V6: 48ul x 95nM = '''456ul''' x 10nM
***Add 385ul H2O
***Add 408ul H2O
**V7: 50ul x 142nM = '''710ul''' x 10nM
**V8: 48ul x 129nM = '''619ul''' x 10nM
***Add 660ul H2O-->
***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====
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''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|650px]]
*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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume (1X)'''
| align="center" style="background:#f0f0f0;"|'''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
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume (1X)'''
| align="center" style="background:#f0f0f0;"|'''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 | 800px]]
 
===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 <br>
~290ul of V8 probes: 183.1 ng/ul => ~ 53 ug
 
===Lambda Exonuclease Digestion===
*Divide each into 6 pcr tubes of 48ul with total amplicon of <10ug each
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| Amplicon||48
|-
| 10X Lambda Exo Buffer||10
|-
| Lambda Exonuclease||10
|-
| H2O||32
|-
| Total||100
|}
 
*Incubated at 37C for 1hr
*Purified with 6 Zymo ssDNA/RNA columns
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
**V6: 64.0ng/ul x 240ul = 15.4ug (67% yield)
**V8: 65.0ng/ul x 240ul = 15.6ug (59% yield)
 
===Remove Amplification Adapters===
====USER====
*Split each set into 3 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 3 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:
  V6: RE DpnII V6
  V8: RE DpnII V8
*Incubated at 94C for 2 min, then 37C for 3 min
*Added 5ul DpnII
*Incubated at 37C for overnight (16 hrs)
 
===TBU Gel: Digestion Check===
*Combine into 1 tube for each set
*2ul Digested Sample + 2ul 2X Urea Loading Dye
*5ul Undigested sample (10nM V8 1st round amplicon) + 5ul 2X Urea Loading Dye
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
[[File:2015-01-28_V6V8_Digest_GelCheck.jpg]]
 
===Zymo Column Purification===
*3 columns for each set
*Eluted 20ul each column (60ul total)
*Nanodrop
**V6: 137.3ng/ul x 60ul = 8.2ug
**V8: 116.2ng/ul x 60ul = 6.9ug
 
===PAGE Size Selection===
*Run 2 gels for V6 and 2 gels for V8
**200V for 35min
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
| V6 Probes||60
|-
| H2O ||60
|-
| TBE-Urea Buffer 2X||120
|}
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
| V8 Probes||60
|-
| H2O ||60
|-
| TBE-Urea Buffer 2X||120
|}
 
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
| Low Mass Ladder||4
|-
| TBE-Urea Buffer 2X||20
|-
| H2O||16
|-
| Total||40
|}
 
===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 8 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 8 tubes at -80C for 30 min
*Spun 8 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 total each)
 
===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
|-
|
|}
*V6
[[File:2015-01-30_V6_GelQuant.jpg|650px]]
*V8
[[File:2015-01-30_V8_GelQuant.jpg|650px]]
*Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear
*V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul)
*V8: 42.7ng/ul =>  ng/ul / (150nt*325Da/nt + 79Da) = 874nM (38ul)
 
[[Media:2015-01-30_V6V7_GelQuant.xlsx | How I calculated concentration]]

Latest revision as of 00:14, 6 February 2015

CA12k_Nov2014 RevComp Probe Production[edit]

Expansion PCR[edit]

  • 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[edit]

  • 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[edit]

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[edit]

  • 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[edit]

  • 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[edit]

  • 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[edit]

  • Divide each into 6 pcr tubes of 48ul with total amplicon of <10ug each
Components Volume
Amplicon 48
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 32
Total 100
  • Incubated at 37C for 1hr
  • Purified with 6 Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • V6: 64.0ng/ul x 240ul = 15.4ug (67% yield)
    • V8: 65.0ng/ul x 240ul = 15.6ug (59% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split each set into 3 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 80
USER 5
Total 85
  • Incubate at 37C for 3 hours

DpnII[edit]

  • Added 15ul of the following
Components Volume
10X DpnII Buffer 10
100uM RE-DpnII guide oligo 5
 RE-DpnII guide oligo:
 V6: RE DpnII V6
 V8: RE DpnII V8
  • Incubated at 94C for 2 min, then 37C for 3 min
  • Added 5ul DpnII
  • Incubated at 37C for overnight (16 hrs)

TBU Gel: Digestion Check[edit]

  • Combine into 1 tube for each set
  • 2ul Digested Sample + 2ul 2X Urea Loading Dye
  • 5ul Undigested sample (10nM V8 1st round amplicon) + 5ul 2X Urea Loading Dye
  • 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye

File:2015-01-28 V6V8 Digest GelCheck.jpg

Zymo Column Purification[edit]

  • 3 columns for each set
  • Eluted 20ul each column (60ul total)
  • Nanodrop
    • V6: 137.3ng/ul x 60ul = 8.2ug
    • V8: 116.2ng/ul x 60ul = 6.9ug

PAGE Size Selection[edit]

  • Run 2 gels for V6 and 2 gels for V8
    • 200V for 35min
Components 2X Volume
V6 Probes 60
H2O 60
TBE-Urea Buffer 2X 120
Components 2X Volume
V8 Probes 60
H2O 60
TBE-Urea Buffer 2X 120
Components Volume
Low Mass Ladder 4
TBE-Urea Buffer 2X 20
H2O 16
Total 40

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 8 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 8 tubes at -80C for 30 min
  • Spun 8 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 total each)

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
  • V6

File:2015-01-30 V6 GelQuant.jpg

  • V8

File:2015-01-30 V8 GelQuant.jpg

  • Only use ladder bands 100bp, 200bp, and 400bp because others were too saturated and non-linear
  • V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul)
  • V8: 42.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 874nM (38ul)

How I calculated concentration