AlanFung:LabNotes/CTCF/2010-4-23

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Revision as of 18:06, 27 April 2010 by >Alan6017518 (→‎TBU Gel Quantification)
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Project Update[edit]

Bottleneck[edit]

  • Extreme low amount of ligated product
  • Low adapter ligation efficiency

Improvements[edit]

  • Start of with high amount of input DNA (500ng & 1ug,50ng)
  • Increase the amount of adapter and increase annealing time

Quantification of Jurkat PCR Product[edit]

Qubit: 48.4ng/ul

Library Prep with new Jurkat PCR product[edit]

  • Concentration:48.4ng/ul
  • BP:100BP
  • setup: 1ug, 500ng and -ve control

End Repair[edit]

1ug 500ng 50ng -ve
DNA 20.66 10.33 1.03 0
End Repair Buffer 10 10 10 10
End Repair Enzyme Mix 5 5 5 5
ddh2o 64.34 74.67 83.97 85
Total Volume 100 100 100 100
  • Incubate at 20C for 30 minutes
  • EtOH purification resolve with 42ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue)

A-Tailing[edit]

1ug 500ng 50ng -ve
End Repaired DNA 42 42 42 42
A-tailing Buffer 5 5 5 5
Klenow Fragment 3 3 3 3
Total Volume 50 50 50 50
  • Incubate at 37C for 30mins
  • EtOH purification resolve with 30ul ddh2o(125ul 100%EtOH, 5ul 3M NaOH, 1ul Glycoblue)

Adapter Ligation[edit]

1ug 500ng 50ng -ve
A-tailed DNA 30 30 30 30
Ligation Buffer 10 10 10 10
Methylation Adapter 5 5 5 5
DNA Ligase 5 5 5 5
Total Volume 50 50 50 50
  • Incubate at 20C for 30mins
  • EtOH purification resolve with 30ul ddh2o

TBU Gel Quantification[edit]

  • 2 ul sample+ 3 ul TBE buffer + 5 ul 2X TBU Buffer
  • 0.5ul LM Ladder + 4.5 ul TBE Buffer + 5 ul 2X TBU Buffer
  • Denature the DNA with loading dye on at 75C for 7 mins, transfer to cooling rack for 1 min
  • Pre run gel

File:ZhangLab 2 2010-04-26 15hr 14min.jpg

Bead Purification[edit]

  • Resolve with 40ul

TBU Gel Quantification[edit]

  • 2 ul sample+ 3 ul TBE buffer + 5 ul 2X TBU Buffer
  • 0.5ul LM Ladder + 4.5 ul TBE Buffer + 5 ul 2X TBU Buffer
  • Denature the DNA with loading dye on at 75C for 7 mins, transfer to cooling rack for 1 min
  • Pre run gel

File:ZhangLab 2 2010-04-26 17hr 47min.jpg

  • Quantify result for 500ng bands
  • 2.3ng-middle band
  • 2.12ng-upperband
  • The amount of DNA loaded to the gel may have overloaded the gel resulting in the big smear in the image
  • amount of ligated product = 40ng