Noi/NOTES/2013-1-15: Difference between revisions
Jump to navigation
Jump to search
>Noi mNo edit summary |
>Noi mNo edit summary |
||
Line 1: | Line 1: | ||
* [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] | * [[http://genome-tech.ucsd.edu/LabNotes/index.php/noi:DMR220k_LabNotes '''Link to calendar''']] | ||
* Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-1-11 | |||
= Skin Microbiom Project = | |||
= Library construction = | = Library construction = | ||
* Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer. | * Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer. |
Revision as of 01:53, 16 January 2013
- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-1-11
Skin Microbiom Project
Library construction
- Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer.
- Note: Lost sample in well A1 (well broken)
- Dr. Zhang suggested to prepare 7 libraries, but keep 3 of them (F1-H1) to be used later and do PCR on 4 samples (B1-E1)
- Estimate concentration by PAGE analysis (to use this amount as the roughly amount of the future experiment by assume that each sample has equal amount of DNA.
File:ZhangLab 2 2013-01-15 12hr 28min purified shearedDNA.jpg
WellID | Conc. (ng/ul) | Yields in 50ul |
B1 | 0.26 | 13.21 |
C1 | 1.60 | 79.87 |
D1 | 3.92 | 196.11 |
E1 | 2.57 | 128.49 |
F1 | 4.67 | 233.58 |
G1 | 1.56 | 78.03 |
H1 | 1.80 | 90.23 |
- From the concentration above, the total amount of amlpicons is not as high as 1ug, but in the range ~20-230ng. Taking the average of the total amount of all 7 samples, it would it ~100ng total.
End repair
Total reaction | 70.00 | ul |
DNA | 50.00 | ul |
End repair reaction mix | 20.00 | ul |
KAPA End repair mix | 1x rxn | 8x rxn mix |
10X End repai buffer | 7.00 | 56.00 |
KAPA End repair enzyme | 5.00 | 40.00 |
H2O | 8.00 | 64.00 |
Total | 20.00 | 160.00 |
- Aliquot to column #9: 22ul
A-tailing
A-tailing reaction mix: Total 30ul/reaction
A-tailing reaction mix | 1x rxn | 8x rxn mix |
KAPA 10X A-tailing buffer | 3.00 | 24.00 |
KAPA A-tailing enzyme | 3.00 | 24.00 |
H2O | 24.00 | 192.00 |
Total | 30.00 | 240.00 |
- Aliquot to column #10: 32ul
Adapter ligation
- Adapter ligation mix: Total 40ul/reaction -> 10ul is adapters
Ligation reaction mix | 1x rxn | 8x rxn mix |
KAPA 5 Ligation buffer | 10.00 | 80.00 |
KAPA DNA ligase | 4.00 | 32.00 |
H2O | 26.00 | 208.00 |
Total | 40.00 | 320.00 |
- Aliquot to column #11: 43ul
Adapter preparation
Assume DNA amount ~100ng, size 400bp MW dA-tailed products = (400bp*607.4 D/bp +157.9 D) = 243.12kD = 243,118 g/mole For 100 ng dA-tailed product = 100ng/243118 g/mole = 4.11E-04 nmole * 20:1 = 0.0082 nmole adapters required. Adapters (ul) = 0.0082nmoles/(20xE3 nmoles/L * 1E-06L/ul) = 0.0082nmoles/(20E-3 nmoles/ul) = 0.411ul I will prepare total volume of 20uM annealed adapters 10ul
Stoffel buffer | 1.00 |
PE_t_adapter | 2.00 |
PE_b_adapter | 2.00 |
H2O | 5.00 |
- 94C 2min -> 0.2C/sec to 20C -> 4C hold
- Dilute annealed adapter with 1x Stoffel buffer 0.41ul to 9.59ul -> anneal adapterr 4.1ul: 1x Stoffelul 95.9
20uM annealed adapter | 4.10 |
10x Stoffel buffer | 10.00 |
H2O | 85.90 |
- Aliquot to column #12: 13ul