AlanFung:LabNotes/CTCF/2010-3-24

From ZhangLabWiki
Jump to navigation Jump to search

Quantification of Jurkat PCR Product[edit]

Nanodrop: 250.7ng/ul
Qubit: 48.4ng/ul
Gel Quant: 18.95ng/ul

File:ZhangLab 2 2010-03-24 11hr 35min.jpg

  • I think both the nanaodrop and gel quantification are biased, for the nanaodrop it gives out an extremely high amount of concentration, this usually happens because nanaodrop cannot differentiate between any contamination. As for the gel image the band at 100bp is much stronger than the ladder band which hits that the concentration is higher than 18.95ng/ul. In this case I would go with the qubit quantification.

Library Prep with new Jurkat PCR product[edit]

  • Concentration:48.4ng/ul
  • BP:100BP
  • setup: 100ng,50ng, 25ng, 10ng, 5ng and -ve control

End Repair[edit]

  • Dilute 10x for 25,10 and 5ng
100ng 50ng 25ng 10ng 5ng -ve
DNA 2.07 1.03 5.17 2.07 1.03 0
End Repair Buffer 10 10 10 10 10 10
End Repair Enzyme mix 5 5 5 5 5 5
ddH2o 82.93 83.97 79.83 82.93 83.97 85
Total Volume 100 100 100 100 100 100
  • Incubate at 20C for 30 minutes
  • EtOH purification resolve with 37ul ddh2o (250ul 100%EtOH, 10ul 3M NaOH, 1ul Glycoblue)

A-Tailing[edit]

100ng 50ng 25ng 10ng 5ng -ve
End Repaired DNA 42 42 42 42 42 42
A-tailing buffer 5 5 5 5 5 5
Klenow Fragment 3 3 3 3 3 3
Total Volume 50 50 50 50 50 50
  • Incubate at 37C for 30mins
  • EtOH purification resolve with 25ul ddh2o

Adapter Ligation[edit]

100ng 50ng 25ng 10ng 5ng -ve
A-tailed DNA 25 25 25 25 25 25
Ligation Buffer 10 10 10 10 10 10
1/10x methylation adapter 10 10 10 10 10 10
DNA Ligase 5 5 5 5 5 5
ddHh2o 50 50 50 50 50 50
  • Incubate at 20C for 20mins
  • EtOH purification resolve with 30ul ddh2o

Gel Quantification[edit]

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