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

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>Mzcai
>Mzcai
 
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Line 168: Line 168:
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**V6: 64.0ng/ul x 240ul = 15.4ug (% yield)
**V6: 64.0ng/ul x 240ul = 15.4ug (67% yield)
**V8: 65.0ng/ul x 240ul = 15.6ug (% yield)
**V8: 65.0ng/ul x 240ul = 15.6ug (59% yield)


===Remove Amplification Adapters===
===Remove Amplification Adapters===
Line 184: Line 184:
| Total||85
| Total||85
|}
|}
*Incubate at 37C for 3 hours<!--
*Incubate at 37C for 3 hours
====DpnII====
====DpnII====
*Added 15ul of the following
*Added 15ul of the following
Line 196: Line 196:
|}
|}
   RE-DpnII guide oligo:
   RE-DpnII guide oligo:
   V4: RE DpnII V4
   V6: RE DpnII V6
   V7: RE DpnII V7
   V8: RE DpnII V8
*Incubated at 94C for 2 min, then 37C for 3 min
*Incubated at 94C for 2 min, then 37C for 3 min
*Added 5ul DpnII
*Added 5ul DpnII
*Incubated at 37C for 4 hr
*Incubated at 37C for overnight (16 hrs)


===TBU Gel: Digestion Check===
===TBU Gel: Digestion Check===
*Combine into 1 tube for each set
*Combine into 1 tube for each set
*2ul Digested Sample + 2ul 2X Urea Loading Dye
*2ul Digested Sample + 2ul 2X Urea Loading Dye
*5ul Undigested sample (10nM 1st round amplicon) + 5ul 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
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
[[File:2015-01-07_V4V7_Digest_GelCheck.jpg]]
[[File:2015-01-28_V6V8_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===
===Zymo Column Purification===
*4 columns for each set
*3 columns for each set
*Eluted 30ul each column (120ul total)
*Eluted 20ul each column (60ul total)
*Nanodrop
*Nanodrop
**V4: 45.0ng/ul x 120ul = 5.4ug
**V6: 137.3ng/ul x 60ul = 8.2ug
**V7: 22.8ng/ul x 120ul = 2.7ug (oddly low but checked with nanodrop twice)
**V8: 116.2ng/ul x 60ul = 6.9ug


===PAGE Size Selection===
===PAGE Size Selection===
*Run 2 gels for V4 and 1 gel for V7
*Run 2 gels for V6 and 2 gels for V8
**220V for 30min
**200V for 35min
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| V4 Probes||120
| V6 Probes||60
|-
| H2O ||60
|-
|-
| TBE-Urea Buffer 2X||120
| TBE-Urea Buffer 2X||120
Line 248: Line 232:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''Components'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
| align="center" style="background:#f0f0f0;"|'''2X Volume'''
|-
|-
| V7 Probes||120
| V8 Probes||60
|-
| H2O ||60
|-
|-
| TBE-Urea Buffer 2X||120
| TBE-Urea Buffer 2X||120
Line 259: Line 245:
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| Low Mass Ladder||3
| Low Mass Ladder||4
|-
|-
| TBE-Urea Buffer 2X||15
| TBE-Urea Buffer 2X||20
|-
|-
| H2O||12
| H2O||16
|-
|-
| Total||30
| Total||40
|}
|}
*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===
===EtOH Precipitation===
Line 282: Line 260:
*Vortexed for 60 min at 37 C in incubator
*Vortexed for 60 min at 37 C in incubator
*Centrifuged at 15,000 rpm for 3 min at RT
*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 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 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)
*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
*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
*Vortexed and placed the 8 tubes at -80C for 30 min
*Spun 6 tubes at 10,000rpm at 4C for 30min
*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 add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min
*Discard supernatant and let dry in hood for 10min
*Discard supernatant and let dry in hood for 10min
*Resuspend each tube with 10ul and recombine (40ul for V4 and 20ul for V7)
*Resuspend each tube with 10ul and recombine (40ul total each)


===TBU Gel Quantification===
===TBU Gel Quantification===
Line 319: Line 297:
|  
|  
|}
|}
[[File:2015-01-09_V4_GelQuant.jpg|650px]]
*V6
[[File:2015-01-09_V7_GelQuant.jpg|650px]]
[[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
*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)
*V6: 41.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 854nM (38ul)
*V7: 6.2 ng/ul => 6.2 ng/ul / (150nt*325Da/nt + 79Da) = 127nM (18ul)
*V8: 42.7ng/ul => ng/ul / (150nt*325Da/nt + 79Da) = 874nM (38ul)


[[Media:2015-01-09_V4V7_GelQuant.xlsx | How I calculated concentration]]-->
[[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