AlanFung:LabNotes/CTCF/2010-4-22: Difference between revisions
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>Alan6017518 (Created page with '==Project Update== ===Bottleneck=== *Extreme low amount of ligated product *Low adapter ligation efficiency ===Improvements=== *Start of with high amount of input DNA (500ng & …') |
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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) | ||
*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. | |||
==A-Tailing== | ==A-Tailing== | ||
{| {{table}} | {| {{table}} |
Revision as of 17:06, 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.
A-Tailing
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
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