AlanFung:LabNotes/CTCF/2010-4-22: Difference between revisions

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*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.  
*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.
*I will repeat the experiment using regular tubes only.
==A-Tailing==
{| {{table}}
| ||1ug||500ng||-ve
|-
| End Repaired DNA||42||42||42
|-
| A-tailing Buffer||5||5||5
|-
| Klenow Fragment ||3||3||3
|-
| total volume||50||50||50
|-
|
|}
*Incubate at 37C for 30mins
*EtOH purification resolve with 25ul ddh2o
==Adapter Ligation==
==Adapter Ligation==
{| {{table}}
{| {{table}}

Revision as of 20:41, 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)
  • 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 -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.

Adapter Ligation

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

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