AlanFung:LabNotes/CTCF/2010-4-22

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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)
  • 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 -ve
DNA 20.66 10.33 0
End Repair Buffer 10 10 10
End Repair Enzyme Mix 5 5 5
ddh2o 64.34 74.67 85
total volume 100 100 100
  • Incubate at 20C for 30 minutes
  • EtOH purification resolve with 42ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue)
  • It appears that EtOH purification is the step that causes the most lost of DNA, I realized that after spinning down of the tube, a blue smear along the tube instead of a well defined pellet is being formed. I did a quick test using a regular .5ml tube instead of the non-stick ones and even without any DNA, a pellet is formed after spinning.
  • I will repeat the experiment using regular tubes only.

Gel Quantification[edit]

File:ZhangLab 2 2010-03-24 21hr 30min.jpg File:ZhangLab 2 2010-03-24 21hr 30min-2.jpg