Revision as of 17:06, 23 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)
- 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