Revision as of 22:58, 17 January 2013 by >Noi
Skin Microbiom Project
Library construction
For details of each step, epMotion log file will be added later. Protocol's name: XXX
- Purify sheared DNA with 1volume AmPURE beads (50ul) and elute with 50ul EB buffer and keep the bead in the well. (Note: since the beads will be reused during library prep, I didn't dilute the beads).
- 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 quantification (to use this amount as the average amount in 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, ~100ng total.
DISCUSSION
(about total DNA amount and problem of small volume DNA transfer to Covaris shearing plate)
XXXX
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
- 30C for 30min
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
- 20C for 15min
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.
20uM adapters (ul) required = 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
- After finish adapter ligation, adapter ligated DNA was resuspended in 15ul EB buffer
Amplification
- TEST if amplification works (in small volume 25ul add 1.25ul of adapter ligated template)
Components
|
1x rxn
|
6x rxn mix
|
adapter ligated DNA |
1.25 |
0.00
|
PCR_F(10uM) |
0.50 |
3.00
|
PCR_R (10uM) |
0.50 |
3.00
|
SYBR Green 50x |
0.10 |
0.60
|
H2O |
10.15 |
60.90
|
Phusion HF, 2xMM |
12.50 |
75.00
|
Total volume |
25.00 |
150.00
|
- Aliquot 23.75ul, add 1.25 of adapter ligated DNA
Program
98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x12, 72C - 2min, hold at 15C
File:QPCR 20130115 TESTpcr.png.png
- PAGE analysis to see the pattern of amplified amplicons (loaded PCR product 2ul, no bead purification)
File:ZhangLab 2 2013-01-15 21hr 12min Test PCR.jpg
- Since PCR worked well, I amplified all adapter ligated DNA (B1-E1, 3 replicates, 5ul of template in 100ul reaction). I reduced number of cycle from 12 to 11 cycles
Components
|
1x rxn
|
13.5rxn mix
|
adapter ligated DNA |
5.00 |
0.00
|
PCR_F(10uM) |
2.00 |
27.00
|
PCR_R.N2IndX(10uM)[X=1-4] |
2.00 |
0.00
|
SYBR Green 50x |
0.80 |
10.80
|
H2O |
40.20 |
542.70
|
Phusion HF, 2xMM |
50.00 |
675.00
|
Total volume |
100.00 |
1350.00
|
- Aliquot 93ul, add 5 of adapter ligated DNA and 2ul of PCR_R.N2Indx
Program
98C - 30s, (98C - 10s, 65C - 20s, 72C - 30s)x11, 72C - 2min, hold at 15C
File:QPCR 20130115 3-replictes B1-E1.png.png
- Note: I used different types of plates (TEST PCR used the real time PCR tube (Eppendort WW), the 3 replicates used the Eppendorf plate for epMotion by set up the back ground as the clear plate)
Position in AC-718-384-well
|
Position in AC-718-96-well_plate1
|
Individual library ID
|
Indx
|
J03 |
B1 |
718_plate1_B1 |
PCR_R.N2Indx1
|
L02 |
C1 |
718_plate1_C1 |
PCR_R.N2Indx2
|
M05 |
D1 |
718_plate1_D1 |
PCR_R.N2Indx3
|
N03 |
E1 |
718_plate1_E1 |
PCR_R.N2Indx4
|
- Pooled 80ul of amplified amplicons from the three replicates (total 240ul) and bead purified (diluted Ampure beads 3X with 20%PEG) with epMotion.
- Eluted with EB buffer total volme 75ul
- Dr. Zhang suggested to pooled 50% of each library and perform PAGE size-selection (may not equal molar ratio).
File:ZhangLab 2 2013-01-17 13hr 37min 718 plate1 B1-E1-PAGE-SS.jpg
- I cut the band at the position between 400-600bp, resuspend sequencing library with H2O total volume 30ul.
- Alan will do qRCR to quantify the library concentration
- Verify the library by PAGE analysis
- Library ID: JG-SkinMB_718_p1_N2_B1-E1_Jan15.2013