Revision as of 00:26, 8 February 2013 by >Noi
Skin Microbiom Project
Library construction (72 samples)
- After DNA shearing on 96-well microtubes, I did bead purification and resuspend DNA in 50ul.
- There were some problem during transfer samples from micro-tubes to 96-well plate. The aluminum foil I used to seal the plate had a very bad quality, since it left the sticky part cover the well after I unsealed it. This block the pipette tip to transfer the liquid. I need to manually make the whole with clean pipette tip to clear the well before transfering sample with the robot. Another problem is the robot didn't completely transfer the liquid to the well (may be I need to adjust the dispensing speed). It have the droplet at the tip and might drop and get contaminate to other wells (I observed this about 3-4 samples)
- I randomly pick 10 samples to run the gel check (I used little amount of beads (saw the bead left in the well very little) but comparable to the previous time, so I kind of worry about if I will lose most of the DNA). Next time I may use undiluted beads instead since it will be re-used in the following steps of library prep.
File:ZhangLab 2 2013-02-07 12hr 45min check shearedDNA aftersheared beadpure.jpg
- From the gem image, I got the intensity of DNA in all 10 samples, and it's pretty close to the rehearsal.
End repair
Total reaction
|
70.00
|
ul
|
DNA |
50.00 |
ul
|
End repair reaction mix |
20.00 |
ul
|
KAPA End repair mix
|
1x rxn
|
75x rxn mix
|
10X End repai buffer |
7.00 |
525.00
|
KAPA End repair enzyme |
5.00 |
375.00
|
H2O |
8.00 |
600.00
|
Total |
20.00 |
1500.00
|
- Aliquot to column #1: 187ul
A-tailing
- A-tailing reaction mix: Total 30ul/reaction
- 30C for 30min
A-tailing reaction mix
|
1x rxn
|
75x rxn mix
|
KAPA 10X A-tailing buffer |
3.00 |
225.00
|
KAPA A-tailing enzyme |
3.00 |
225.00
|
H2O |
24.00 |
1800.00
|
Total |
30.00 |
2250.00
|
- Aliquot to column #2 (for 40rxn): 155ul, column #3 (for 32 rxn): 124ul
Adapter ligation
- Adapter ligation mix (Total 50ul) : Ligation reaction mix 40ul/reaction & 10ul of adapters
- 20C for 15min
Ligation reaction mix
|
1x rxn
|
75x rxn mix
|
KAPA 5X Ligation buffer |
10.00 |
750.00
|
KAPA DNA ligase |
3.00 |
225.00
|
H2O |
27.00 |
2025.00
|
Total |
40.00 |
3000.00
|
- Aliquot to column #4 (for 40rxn): 207ul, column #5 (for 32 rxn): 165ul
Adapter preparation
Assume DNA amount ~100ng, size 400bp
MW dA-tailed products = (400bp*607.4 D/bp +157.9 D) = 243.12kD = 243118 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-6L/ul) = 0.0082nmoles/ (20E-3 nmoles/ul) = 0.411ul
- I will prepare total volume of 20uM annealed adapters 40ul
Components
|
10ul
|
40ul
|
Stoffel buffer |
1.00 |
4.00
|
PE_t_adapter |
2.00 |
8.00
|
PE_b_adapter |
2.00 |
8.00
|
H2O |
5.00 |
20.00
|
- 94C 2min -> 0.2C/sec to 20C -> 4C hold
- Dilute annealed adapter with 1x Stoffel buffer 0.41ul to 9.59ul -> I will prepare diluted adapters for ~90rxn)
20uM annealed adapter |
4.10 (for 10 rxn |
36.90 (for ~90 rxn)
|
10x Stoffel buffer |
10.00 |
90.00
|
H2O |
85.90 |
773.10
|
Total |
100.00 |
900.00
|
- Aliquot to column #6 = 112ul