Noi/NOTES/2013-1-15

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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