AlanFung:LabNotes/CTCF/2010-4-23
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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,50ng)
- 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 | 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[edit]
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 30ul ddh2o(125ul 100%EtOH, 5ul 3M NaOH, 1ul Glycoblue)
Adapter Ligation[edit]
1ug | 500ng | 50ng | -ve | |
A-tailed DNA | 30 | 30 | 30 | 30 |
Ligation Buffer | 10 | 10 | 10 | 10 |
Methylation Adapter | 5 | 5 | 5 | 5 |
DNA Ligase | 5 | 5 | 5 | 5 |
Total Volume | 50 | 50 | 50 | 50 |
- Incubate at 20C for 30mins
- EtOH purification resolve with 30ul ddh2o
TBU Gel Quantification[edit]
- 2 ul sample+ 3 ul TBE buffer + 5 ul 2X TBU Buffer
- 0.5ul LM Ladder + 4.5 ul TBE Buffer + 5 ul 2X TBU Buffer
- Denature the DNA with loading dye on at 75C for 7 mins, transfer to cooling rack for 1 min
- Pre run gel
File:ZhangLab 2 2010-04-26 15hr 14min.jpg
Bead Purification[edit]
- Resolve with 40ul
TBU Gel Quantification[edit]
- 2 ul sample+ 3 ul TBE buffer + 5 ul 2X TBU Buffer
- 0.5ul LM Ladder + 4.5 ul TBE Buffer + 5 ul 2X TBU Buffer
- Denature the DNA with loading dye on at 75C for 7 mins, transfer to cooling rack for 1 min
- Pre run gel
File:ZhangLab 2 2010-04-26 17hr 47min.jpg
- Quantify result for 500ng bands
- 2.3ng-middle band
- 2.12ng-upperband
- The amount of DNA loaded to the gel may have overloaded the gel resulting in the big smear in the image
- amount of ligated product = 40ng