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
mNo edit summary
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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]]
*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:2015-01-06_1stRndAmplicon_V4V7_Gel.jpg]]
 
===Production PCR===
*V4 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_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
{| {{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_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 | 800px]]
 
===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
{| {{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]]-->

Revision as of 22:27, 26 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

  • 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