AlanFung:LabNotes/CTCF/2010-4-23: Difference between revisions

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>Alan6017518
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*Incubate at 20C for 30 minutes
*Incubate at 20C for 30 minutes
*EtOH purification resolve with 42ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue)
*EtOH purification resolve with 42ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue)
==A-Tailing==
{| {{table}}
| ||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 25ul ddh2o

Revision as of 20:42, 23 April 2010

Project Update

Bottleneck

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

Improvements

  • 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

Qubit: 48.4ng/ul

Library Prep with new Jurkat PCR product

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

End Repair

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

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 25ul ddh2o