Matt:LabNotes/2015-1-15: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>Mzcai
>Mzcai
 
(11 intermediate revisions by the same user not shown)
Line 87: Line 87:
*Eluted with 40ul each
*Eluted with 40ul each
*Recombined and measured ssDNA with Nanodrop:
*Recombined and measured ssDNA with Nanodrop:
**V4: 77.0ng/ul x 120ul = 9.24ug (% yield)
**V4: 77.0ng/ul x 120ul = 9.24ug (78.6% yield)
**V7: 61.0ng/ul x 120ul = 7.32ug (% yield)
**V7: 61.0ng/ul x 120ul = 7.32ug (59.8% yield)
<!--
 
===Remove Amplification Adapters===
===Remove Amplification Adapters===
====USER====
====USER====
Line 97: Line 97:
| align="center" style="background:#f0f0f0;"|'''Volume'''
| align="center" style="background:#f0f0f0;"|'''Volume'''
|-
|-
| SS-amplicon||80
| SS-amplicon||60
|-
|-
| USER||5
| USER||5
|-
| H2O||20
|-
|-
| Total||85
| Total||85
Line 124: Line 126:
*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 V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
*0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
*2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye)
[[File:2015-01-07_V4V7_Digest_GelCheck.jpg]]
[[File:2015-01-20_V4V7_Digest_GelCheck.jpg]]
*Digested samples (lanes 3 & 5) show significant middle bands indicating incomplete removal of amplification adapters
*V7 Undigested (middle lane) is extremely faint but can see band around 200bp
*To get a higher yield I continued digestion overnight
*Not sure why V4 lane is squiggly but can see the 3 bands
*V7 lane shows only 2 bands but both are below the undigested V7 band


===Zymo Column Purification===
===Zymo Column Purification===
*4 columns for each set
*2 columns for each set
*Eluted 30ul each column (120ul total)
*Eluted 30ul each column (60ul total)
*Nanodrop
*Nanodrop
**V4: 45.0ng/ul x 120ul = 5.4ug
**V4: 78.8ng/ul x 60ul = 4.7ug
**V7: 22.8ng/ul x 120ul = 2.7ug (oddly low but checked with nanodrop twice)
**V7: 38.8ng/ul x 60ul = 2.3ug


===PAGE Size Selection===
===PAGE Size Selection===
*Run 2 gels for V4 and 1 gel for V7
*Run 2 gels for V4 and 1 gel for V7
**220V for 30min
**200V for 30min
{| {{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
| V4 Probes||60
|-
|-
| TBE-Urea Buffer 2X||120
| TBE-Urea Buffer 2X||60
|}
|}


Line 153: Line 156:
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
| align="center" style="background:#f0f0f0;"|'''1X Volume'''
|-
|-
| V7 Probes||120
| V7 Probes||60
|-
|-
| TBE-Urea Buffer 2X||120
| TBE-Urea Buffer 2X||60
|}
|}


Line 172: Line 175:


*V4 Gel 1
*V4 Gel 1
[[File:2015-01-08_SizeSelect_V4_Gel1.jpg]]
[[File:2015-01-20_SizeSelect_V4_Gel1.jpg]]
*V4 Gel 2
*V4 Gel 2
[[File:2015-01-08_SizeSelect_V4_Gel2.jpg]]
[[File:2015-01-20_SizeSelect_V4_Gel2.jpg]]
*V7 Gel 1
*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:2015-01-20_SizeSelect_V7_Gel1.jpg]]
[[File:2015-01-08_SizeSelect_V7_Gel1.jpg]]


===EtOH Precipitation===
===EtOH Precipitation===
Line 187: Line 189:
*Transfered the clear spnt. to 6 Nanosep column and centrifuged at 15,000 rpm for 3 min
*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 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 (~450ul per tube except for 1 V4 tube which spilled)
*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, 45ul of 3M NaoAc pH 5.2
*Vortexed and placed the 6 tubes at -80C for overnight
*Vortexed and placed the 6 tubes at -80C for overnight
*Spun 6 tubes at 10,000rpm at 4C for 30min
*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 add 650ul cold 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 for V4 and 20ul for V7)
Line 222: Line 224:
|  
|  
|}
|}
[[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]]
*V4
-->
**2ul sample lane was loaded poorly so was not used in concentration calculations
[[File:2015-01-24_V4_GelQuant.jpg|650px]]
 
*V7
**Only use ladder bands 100bp, 200bp, and 400bp
[[File:2015-01-21_V7_GelQuant.jpg|650px]]
 
*V4: 65.3 ng/ul => 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul)
*V7: 39.2 ng/ul => 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul)
 
[[Media:2015-01-21_V7_GelQuant.xlsx | V7 Concentration calculation]]
[[Media:2015-01-24_V4_GelQuant.xlsx | V4 Concentration calculation]]

Latest revision as of 22:34, 24 January 2015

CA12k_Nov2014 V4 and V7 Probe Production[edit]

Production PCR[edit]

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 15sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold [[File: | 800px]]

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 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 50ul H2O

Qia Column Purification[edit]

  • Purified in 6 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:

~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug ~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug

Lambda Exonuclease Digestion[edit]

  • Divide each into 3 pcr tubes of 50ul with total amplicon of <10ug each
Components Volume
Amplicon 50
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 30
Total 100
  • Incubated at 37C for 1hr
  • Purified with 3 Zymo ssDNA/RNA columns each
  • Eluted with 40ul each
  • Recombined and measured ssDNA with Nanodrop:
    • V4: 77.0ng/ul x 120ul = 9.24ug (78.6% yield)
    • V7: 61.0ng/ul x 120ul = 7.32ug (59.8% yield)

Remove Amplification Adapters[edit]

USER[edit]

  • Split each set into 2 PCR tubes and add 5ul USER
Components Volume
SS-amplicon 60
USER 5
H2O 20
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 overnight

TBU Gel: Digestion Check[edit]

  • Combine into 1 tube for each set
  • 2ul Digested Sample + 2ul 2X Urea Loading Dye
  • 5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
  • 2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye)

File:2015-01-20 V4V7 Digest GelCheck.jpg

  • V7 Undigested (middle lane) is extremely faint but can see band around 200bp
  • Not sure why V4 lane is squiggly but can see the 3 bands
  • V7 lane shows only 2 bands but both are below the undigested V7 band

Zymo Column Purification[edit]

  • 2 columns for each set
  • Eluted 30ul each column (60ul total)
  • Nanodrop
    • V4: 78.8ng/ul x 60ul = 4.7ug
    • V7: 38.8ng/ul x 60ul = 2.3ug

PAGE Size Selection[edit]

  • Run 2 gels for V4 and 1 gel for V7
    • 200V for 30min
Components 2X Volume
V4 Probes 60
TBE-Urea Buffer 2X 60
Components 1X Volume
V7 Probes 60
TBE-Urea Buffer 2X 60
Components Volume
Low Mass Ladder 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • V4 Gel 1

File:2015-01-20 SizeSelect V4 Gel1.jpg

  • V4 Gel 2

File:2015-01-20 SizeSelect V4 Gel2.jpg

  • V7 Gel 1

File:2015-01-20 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 (~450ul per tube except for 1 V4 tube which spilled)
  • Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul 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 cold 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
  • V4
    • 2ul sample lane was loaded poorly so was not used in concentration calculations

File:2015-01-24 V4 GelQuant.jpg

  • V7
    • Only use ladder bands 100bp, 200bp, and 400bp

File:2015-01-21 V7 GelQuant.jpg

  • V4: 65.3 ng/ul => 65.3 ng/ul / (150nt*325Da/nt + 79Da) = 1337nM (38ul)
  • V7: 39.2 ng/ul => 39.2 ng/ul / (150nt*325Da/nt + 79Da) = 803nM (18ul)

V7 Concentration calculation V4 Concentration calculation