Noi/NOTES/2013-1-15
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- [Link to calendar]
- Continued from: http://genome-tech.ucsd.edu/LabNotes/index.php/Noi/NOTES/2013-1-11
Skin Microbiom Project[edit]
Library construction[edit]
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[edit]
(about total DNA amount and problem of small volume DNA transfer to Covaris shearing plate)
XXXX
End repair[edit]
Total reaction | 70.00 | ul |
DNA | 50.00 | ul |
End repair reaction mix | 20.00 | ul |
- 20C for 30min
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[edit]
- 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[edit]
- 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[edit]
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[edit]
- 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
File:ZhangLab 2 2013-01-17 16hr 07min SkinMB 718 p1 N2 B1-E1 Jan15-e.jpg
- Library ID: JG-SkinMB_718_p1_N2_B1-E1_Jan15.2013
- I performed PAGE quantification on individual library (B1-E1) in the range 400-600bp for further experiment (DSN)
Position in AC-718-384-well |
Position in AC-718-96-well_plate1 |
Individual library ID | Conc. (ng/ul) | Yields in 35ul (ng) |
J03 | B1 | 718_plate1_B1 | 0.88 | 30.86 |
L02 | C1 | 718_plate1_C1 | 2.59 | 90.55 |
M05 | D1 | 718_plate1_D1 | 3.12 | 109.08 |
N03 | E1 | 718_plate1_E1 | 2.74 | 95.82 |