Noi/NOTES/2013-1-15

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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
  • 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

  • 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

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) on the band 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