Revision as of 17:24, 27 April 2010 by >Alan6017518
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 30ul ddh2o(125ul 100%EtOH, 5ul 3M NaOH, 1ul Glycoblue)
Adapter Ligation
|
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
- 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
TBU Gel Quantification
- 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