Matt:LabNotes/2013-5-17: Difference between revisions

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>Mzcai
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>Mzcai
 
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Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-16
Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-16


===PAGE-Size Selection of 0 gap probes===
===PAGE-Size Selection of 20 gap probes===


*Noi said that the band on the previous TBU gel looked relatively faint and recommended putting 3ug of DNA per well instead of the usual 2ug to increase yield
*Noi said that the band on the previous TBU gel looked relatively faint and recommended putting 3ug of DNA per well instead of the usual 2ug to increase yield
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| align="center" style="background:#f0f0f0;"|'''3X Volume'''
| align="center" style="background:#f0f0f0;"|'''3X Volume'''
|-
|-
| Enzyme digested probes (~10ug/180ul||60||180
| Enzyme digested probes (~10ug/180ul)||55||165
|-
|-
| TBE-Urea Buffer 2X||60||180
| TBE-Urea Buffer 2X||55||165
|-
|-
| Water||0||0
| Water||0||0
|-
|-
| Total||120||360
| Total||110||330
|-
|-
|  
|  
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{| {{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;"|'''3X Volume'''
|-
|-
| Low Mass DNA ladder (1 ug/ul)||3
| Low Mass DNA ladder (1 ug/ul)||3
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|  
|  
|}
|}
*Ran the 3 gels at 220V for 30min
*Cut out band below 200bp
Gel 1: <br>
[[File:2013-05-17_Gel1SizeSelection_20gap.jpg| 450px]]
Gel 2: <br>
[[File:2013-05-17_Gel2SizeSelection_20gap.jpg| 450px]]
Gel 3: <br>
[[File:2013-05-17_Gel3SizeSelection_20gap.jpg| 450px]]
*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 1.5 mL tube below with pipette tip.
*Added 450 ul of 1X TE buffer
*Vortexed for 45-60 min at 37 C in incubator
*Centrifuged at 15,000 rpm for 3 min at RT
*Transfered the clear spnt. to 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 (volume ~ 400ul/ 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 overnight
Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-21

Latest revision as of 00:13, 27 June 2013

Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-16

PAGE-Size Selection of 20 gap probes[edit]

  • Noi said that the band on the previous TBU gel looked relatively faint and recommended putting 3ug of DNA per well instead of the usual 2ug to increase yield
  • I will run 3 gels each with 60ul (~3.4 ug of DNA each)

Sample

Components 1X Volume 3X Volume
Enzyme digested probes (~10ug/180ul) 55 165
TBE-Urea Buffer 2X 55 165
Water 0 0
Total 110 330

3 Low Mass DNA Ladders

Components 3X Volume
Low Mass DNA ladder (1 ug/ul) 3
TBE-Urea Buffer 2X 15
H2O 12
Total 30
  • Ran the 3 gels at 220V for 30min
  • Cut out band below 200bp

Gel 1:
File:2013-05-17 Gel1SizeSelection 20gap.jpg

Gel 2:
File:2013-05-17 Gel2SizeSelection 20gap.jpg

Gel 3:
File:2013-05-17 Gel3SizeSelection 20gap.jpg

  • 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 1.5 mL tube below with pipette tip.
  • Added 450 ul of 1X TE buffer
  • Vortexed for 45-60 min at 37 C in incubator
  • Centrifuged at 15,000 rpm for 3 min at RT
  • Transfered the clear spnt. to 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 (volume ~ 400ul/ 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 overnight


Continued on: http://genome-tech.ucsd.edu/LabNotes/index.php/Matt:LabNotes/2013-5-21